Cfdna standard and preparation method therefor

Abstract

The present invention relates to a cfDNA standard and a preparation method therefor. The method comprises the following steps: a1) constructing a vector expressing DNA fragmentation factor subunit beta (DFFB) or deoxyribonuclease 1 like 3 (DNASE1L3); a2) culturing cells to be transfected; b) transfecting a vector having a DFFB gene and a vector having a DNASE1L3 gene into said cells and culturing same; and c) for the successfully transfected cells, collecting culture supernatant and extracting cfDNA.

Description

cfDNA Standards and Their Preparation Methods Technical Field

[0001] This invention relates to the field of high-throughput sequencing, specifically to a method for preparing cell-free DNA (cfDNA) standards and the cfDNA standards obtained therefrom. Background Technology

[0002] High-throughput sequencing technology for detecting cfDNA methylation in blood is being used more and more widely in the field of early cancer screening. Although this technology has a promising future, it is still under development and being gradually improved. The development and establishment of this technology requires a large number of samples as experimental materials. However, real sample sources are relatively precious and the cost and difficulty of obtaining them are high.

[0003] Currently, mechanically fragmented genomic DNA (gDNA) fragments are commonly used as cfDNA standards for high-throughput sequencing to detect methylation of cfDNA in blood. However, due to the randomness of mechanical fragmentation, the sequence information, fragment length, and other characteristics of the DNA fragments obtained by this method differ significantly from natural cfDNA. Furthermore, existing technologies also use MNase and Atlantis dsDNase to digest gDNA in vitro to obtain cfDNA standards; however, these enzymes are derived from bacteria, and the cfDNA standards obtained by digesting human gDNA with these enzymes also differ significantly from natural cfDNA.

[0004] In addition, all of the above-mentioned existing methods require prior extraction of gDNA, which makes the operation more complicated; on the other hand, the quality of the extracted gDNA will also affect the quality of the cfDNA standard prepared subsequently.

[0005] Therefore, there is an urgent need in the field for a preparation method that can produce cfDNA standards that are less different from natural cfDNA and is simpler to operate, as well as cfDNA standards that are less different from natural cfDNA. Summary of the Invention

[0006] This invention provides a method for preparing cfDNA standards and the cfDNA standards obtained therefrom, which are less different from natural cfDNA, and the preparation method is simpler to operate.

[0007] In one aspect, the present invention provides a method for preparing cfDNA standards, comprising the following steps:

[0008] a1) Construct vectors expressing DFFB (DNA Fragmentation Factor subunit Beta) and / or DNASE1L3 (deoxyribonuclease 1 like 3);

[0009] a2) Culture the cells to be transfected;

[0010] b) Transfect cells with a vector carrying the DFFB gene and / or a vector carrying the DNASE1L3 gene and culture them.

[0011] c) For successfully transfected cells, collect the culture medium supernatant and extract DNA. The obtained DNA is the cfDNA standard.

[0012] In one implementation, in step a1), the DFFB gene and the DNASE1L3 gene are located on the same vector or on different vectors.

[0013] In a preferred embodiment, in step b), the vector carrying the DFFB gene and the vector carrying the DNASE1L3 gene are co-transfected into the cells and cultured.

[0014] In one embodiment, the vector for expressing DFFB and / or the vector for expressing DNASE1L3 are mammalian gene expression vectors.

[0015] In one embodiment, the vector used to express DFFB and / or the vector used to express DNASE1L3 is a plasmid vector or a viral vector.

[0016] In one embodiment, the vector for expressing DFFB is the plasmid shown in sequence 1, and / or the vector for expressing DNASE1L3 is the plasmid shown in sequence 2.

[0017] In one implementation, the DFFB gene (e.g., as shown in sequence: 3) is synthesized artificially or isolated from human, and / or the DNASE1L3 gene (e.g., as shown in sequence: 4) is synthesized artificially or isolated from human.

[0018] In one embodiment, the DFFB gene is ligated with the plasmid shown in sequence 1 to form the plasmid shown in sequence 5, and / or the DNASE1L3 gene is ligated with the plasmid shown in sequence 2 to form the plasmid shown in sequence 6.

[0019] In one implementation, in step a2), when the cells are cultured to the logarithmic growth phase, they are passaged.

[0020] In one embodiment, the cells are adherent cells (e.g., cells derived from liver cancer cell lines or colon cancer cell lines) or suspension cells (e.g., cells derived from lymphocyte cell lines).

[0021] In one implementation, during step a2), when passaged, the cells are cultured to a confluence of 50-80% (e.g., 50, 55, 60, 65, 70, 75, or 80%, or a subrange of any value in these ranges).

[0022] In one embodiment, during the transfection process in step b), the total amount of the vector carrying the DFFB gene and / or the vector carrying the DNASE1L3 gene is 1-6 μg, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 μg, or a subrange consisting of any values ​​within these ranges. In a preferred embodiment, during the transfection process in step b), the total amount of the vector carrying the DFFB gene and the vector carrying the DNASE1L3 gene is 1-6 μg, and the mass ratio of the vector carrying the DFFB gene to the vector carrying the DNASE1L3 gene is (1-2):(1-2), for example, 1:1, 1:2, 1.5:1, 1.5:2, 2:1, 1:1.5, or 2:1.5. Most preferably, the mass ratio of the vector carrying the DFFB gene to the vector carrying the DNASE1L3 gene is 1:1.

[0023] In one embodiment, during the transfection process in step b), the vector carrying the DFFB gene and / or the vector carrying the DNASE1L3 gene are mixed with 100-300 μL of culture medium (e.g., 100, 110, 120, 130, 140, 150, 200, 250 or 300 μL, or a subrange of any value in these ranges) to obtain a first mixture.

[0024] In one embodiment, the resulting first mixture is centrifuged at 500-1500 rpm (e.g., 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 rpm, or a subrange of any value in these ranges) for 15-45 s (e.g., 15, 20, 25, 30, 35, 40 or 45 s, or a subrange of any value in these ranges).

[0025] In one embodiment, the resulting first mixture is centrifuged and then allowed to stand for at least 5 minutes, for example, at least 5, 6, 7, 8, 9 or 10 minutes, or a subrange consisting of any values ​​within these ranges.

[0026] In one embodiment, during the transfection process in step b), the transfection reagent used is 2-12 μL, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 μL, or a subrange consisting of any values ​​within these ranges.

[0027] In one embodiment, during the transfection process in step b), the transfection reagent is mixed with 100-300 μL of culture medium (e.g., 100, 110, 120, 130, 140, 150, 200, 250 or 300 μL, or a subrange of any value in these ranges) to obtain a second mixture.

[0028] In one embodiment, the resulting second mixture is centrifuged at 500-1500 rpm (e.g., 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 rpm, or a subrange of any value in these ranges) for 15-45 s (e.g., 15, 20, 25, 30, 35, 40 or 45 s, or a subrange of any value in these ranges).

[0029] In one embodiment, the resulting second mixture is centrifuged and then allowed to stand for at least 5 minutes, for example, at least 5, 6, 7, 8, 9 or 10 minutes, or a subrange consisting of any values ​​within these ranges.

[0030] In one embodiment, the first mixture and the second mixture are mixed and then added to a container culturing cells to be transfected.

[0031] In one implementation, after transfection in step b), the cells are cultured for 12-60 h, for example, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 h, or a subrange consisting of any values ​​in these ranges.

[0032] In one embodiment, the culture medium may be a complete culture medium or a minimum required culture medium.

[0033] In one embodiment, the complete culture medium can be a basal medium containing fetal bovine serum, for example, the basal medium can be various existing basal media such as DMEM basal medium, McCoy's 5A basal medium, or RPMI 1640 basal medium. Preferably, the proportion of fetal bovine serum is 8-12% (v / v), for example, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12% (v / v).

[0034] In one embodiment, the minimum required culture medium is a serum-depleted culture medium, such as Opti-MEM. TM Serum-depleted culture medium.

[0035] In one implementation, in step c), the success of transfection is determined based on the expression of the marker gene.

[0036] In one implementation, in step c), at least 1 mL of the culture supernatant of the successfully transfected cells is collected, for example, at least 1, 2, 3, 4 or 5 mL, or a subrange consisting of any values ​​in these ranges.

[0037] In one embodiment, the supernatant is centrifuged to remove residual cells; preferably, centrifuged at 4000-5000 rpm (e.g., 4000, 4100, 4200, 4300, 4500, 4600, 4700, 4800, 4900 or 5000 rpm, or a subrange of any value in these ranges) for at least 4 min (e.g., at least 4, 5, 6, 7, 8, 9 or 10 min, or a subrange of any value in these ranges).

[0038] In one embodiment, the supernatant is subjected to cfDNA extraction. For example, this can be done using any available commercial kit.

[0039] In another aspect, the present invention provides a cfDNA standard, which is prepared by the method for preparing cfDNA standards of the present invention.

[0040] The transfection reagents in this invention may include any available reagents or combinations thereof that facilitate the transfection of a vector into cells, such as liposomes, calcium phosphate, polyethyleneimine (PEI), or any combination thereof.

[0041] All numerical values ​​in this invention are approximate values ​​after rounding.

[0042] Unlike traditional methods that extract gDNA and then directly digest it in vitro with commercially available enzymes to obtain cfDNA standards, this invention uniquely transforms vectors expressing DFFB and / or DNASE1L3 into corresponding in vitro cultured cells, obtaining cfDNA standards from the supernatant that are more similar to natural cfDNA and meet the required quantities. This method is contrary to the prior knowledge of those skilled in the art. Those skilled in the art generally believe that:

[0043] First, in living cells, gDNA is typically located within the nuclear membrane. Even after transfecting cells with a vector capable of expressing the corresponding enzyme, and despite successful enzyme expression, the enzyme cannot interact with the gDNA. Even during the cell cycle phase where the nuclear membrane periodically disappears, the gDNA is protected by histones and remains highly coiled, preventing effective enzyme contact. Even assuming a partial reaction occurs at this stage, enzyme cleavage of the gDNA would severely damage it, leading to apoptosis and disrupting continuous cell production.

[0044] Secondly, the conditions for cell culture and transfection are quite demanding, and the low success rate of transfection and expression will prevent those skilled in the art from obtaining cfDNA standards by transfecting vectors that express the corresponding enzymes into living cells. Instead, they will use a seemingly more convenient method of extracting gDNA and then directly digesting it in vitro with the corresponding commercial enzymes to obtain cfDNA standards.

[0045] Furthermore, even if it is believed that the enzyme can be secreted into the culture medium to cleave gDNA after being expressed in the cell, the low gDNA content in the culture medium and the complex environment of the culture medium will cause those skilled in the art to abandon the method in this invention due to concerns about the yield and whether the enzyme can achieve good activity in the culture system, and instead use the traditional method of extracting gDNA and then digesting it with enzymes, which has more controllable yield and enzyme action environment.

[0046] However, the inventors unexpectedly discovered that after transfecting cells with vectors expressing DFFB and / or DNASE1L3 using the method of the present invention and culturing them for a certain period of time, the cells were in good condition, and sufficient DNA fragments suitable for use as cfDNA standards were found in the supernatant, which were less different from natural cfDNA.

[0047] This invention eliminates the traditional steps of gDNA extraction, allowing direct extraction of cfDNA standards from the supernatant. The process is simpler and suitable for continuous industrial production. Furthermore, the resulting cfDNA standards show less difference from natural cfDNA. Attached Figure Description

[0048] Figure 1A shows the cell state after transfection with the empty vector plasmid in Comparative Example 1; Figure 1B shows the peak shape of the cfDNA standard prepared in Comparative Example 1. The horizontal axis represents fragment size, and the vertical axis represents fluorescence intensity.

[0049] Figure 2A shows the cell state after transfection with the plasmid in Example 1; Figure 2B shows the peak shape of the cfDNA standard prepared in Example 1. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0050] Figure 3A shows the cell state after transfection with the plasmid in Example 2; Figure 3B shows the peak shape of the cfDNA standard prepared in Example 2. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0051] Figure 4A shows the cell state after transfection with the empty vector plasmid in Comparative Example 2; Figure 4B shows the peak shape of the cfDNA standard prepared in Comparative Example 2. The horizontal axis represents fragment size, and the vertical axis represents fluorescence intensity.

[0052] Figure 5A shows the cell state after transfection with the plasmid in Example 3; Figure 5B shows the peak shape of the cfDNA standard prepared in Example 3. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0053] Figure 6A shows the cell state after transfection with the plasmid in Example 4; Figure 6B shows the peak shape of the cfDNA standard prepared in Example 4. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0054] Figure 7A shows the cell state after transfection with the empty vector plasmid in Comparative Example 5; Figure 7B shows the peak shape of the cfDNA standard prepared in Comparative Example 5. The horizontal axis represents fragment size, and the vertical axis represents fluorescence intensity.

[0055] Figure 8A shows the cell state after transfection with the plasmid in Example 5; Figure 8B shows the peak shape of the cfDNA standard prepared in Example 5. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0056] Figure 9A shows the cell state after transfection with the plasmid in Example 6; Figure 9B shows the peak shape of the cfDNA standard prepared in Example 6. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0057] Figure 10A shows the cell state after transfection with the empty vector plasmid in Example 7; Figure 10B shows the peak shape of the cfDNA standard prepared in Example 7. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0058] Figure 11 shows the distribution of cfDNA standard library fragments prepared in the embodiments and comparative examples of the present invention. The horizontal axis represents fragment size, and the vertical axis represents fluorescence value.

[0059] Figure 12 shows a comparison of GC bias between the cfDNA standards prepared in the embodiments of the present invention and those prepared in the comparative examples. The horizontal axis represents GC content, and the vertical axis represents the normalized sequencing depth after Lowess regression smoothing. Detailed Implementation

[0060] To better understand the present invention, the following embodiments further illustrate the content of the invention, but the content of the invention is not limited to the following embodiments. Unless otherwise specified, the experimental operations described in the following embodiments are routine operations; the reagents and materials described are commercially available unless otherwise specified.

[0061] General Embodiments

[0062] 1. Cell culture (select culture method 1.1 or 1.2 according to cell type)

[0063] 1.1 Adherent cell culture

[0064] a. Before the experiment, remove the complete culture medium, trypsin, and PBS from the refrigerator and preheat them to 37°C;

[0065] b. Wipe the outside of the culture medium bottles, trypsin bottles, and PBS bottles with alcohol swabs and place them in the work area;

[0066] c. Remove the culture dish from the cell culture incubator and observe the cell growth under an inverted microscope beforehand. When the cells are in the logarithmic growth phase, passage them.

[0067] d. Place the petri dish on the worktable and use a suction pump to remove the original culture medium;

[0068] e. Slowly add 1 mL of PBS along the sidewall of the petri dish to wash twice;

[0069] f. Add 1 mL of trypsin and spread it evenly on the bottom of the culture dish, then aspirate and discard it. Incubate in an incubator (37℃) for 2-4 min to digest.

[0070] g. Add 1 mL of complete culture medium to stop digestion, gently pipette the cells from the bottom, and collect the cells into a 15 mL centrifuge tube;

[0071] h. Place the centrifuge tubes in the centrifuge and centrifuge at 1000 rpm for 4 minutes;

[0072] i. Resuspend the cells in 1 mL of complete culture medium, count the cells, and perform a (1-2)*10⁻⁶ cell count. 6 Seed cells per well in a 6-well plate and add culture medium to 2 mL / well. Incubate at 37°C, 5 (v / v)% CO2 for 1-3 days, until cells reach 50-80% confluence.

[0073] 1.2 Suspension Cell Culture

[0074] a. Before the experiment, remove the complete culture medium from the refrigerator and preheat it to 37°C;

[0075] b. Wipe the outside of the culture medium bottle with an alcohol swab and place it on the workbench;

[0076] c. Remove the culture dish from the cell culture incubator and observe the cell growth under an inverted microscope beforehand. When the cells are in the logarithmic growth phase, passage them.

[0077] d. Place the culture dish on the operating table and use a pipette to transfer the cells into a 15mL centrifuge tube;

[0078] e. Place the centrifuge tubes in the centrifuge and centrifuge at 1000 rpm for 4 minutes;

[0079] i. Resuspend the cells in 1 mL of complete culture medium, count the cells, and perform a cell count according to (0.5-0.8)*10⁻⁶. 6 Seed cells per well in a 6-well plate and add culture medium to 2 mL / well. Incubate at 37°C, 5 (v / v)% CO2 for 1-3 days, until cells reach 50-80% confluence.

[0080] 2. Plasmid construction

[0081] The DFFB gene (as shown in sequence 3) and the DNASE1L3 gene (as shown in sequence 4) were artificially synthesized by a third-party institution. The DFFB gene was then constructed into the plasmid shown in sequence 1 to obtain the plasmid shown in sequence 5; the DNASE1L3 gene was constructed into the plasmid shown in sequence 2 to obtain the plasmid shown in sequence 6.

[0082] 3. Plasmid transfection

[0083] a. Before the experiment, Opti-MEM TM Remove the serum-depleted culture medium, Lipofectamine 3000 transfection reagent, and plasmid from the refrigerator and allow them to return to room temperature.

[0084] b. Remove the culture dish from the cell culture incubator and observe the cell growth under an inverted microscope beforehand. When the cells reach 50-80% confluence, transfect them.

[0085] c. Wipe the culture medium bottles, transfection reagents, and plasmid packaging tubes with alcohol swabs and place them on the operating table;

[0086] d. Add 1-6 μg of plasmid (where the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1) to 100-300 μL of Opti-MEM. TM Mix the serum-reduced culture medium, centrifuge briefly at low speed (1000 rpm for 30 seconds), and let stand for 5 minutes.

[0087] e. Add 2-12 μL of transfection reagent to 100-300 μL of Opti-MEM. TMMix the serum-reduced culture medium, centrifuge briefly at low speed (1000 rpm for 30 seconds), and let stand for 5 minutes.

[0088] f. Add the mixed transfection reagent to the mixed plasmid, gently pipette 15 times with a 200 μL pipette tip to mix, centrifuge briefly at low speed (1000 rpm for 30 s), and let stand for 15-25 min.

[0089] g. Add the mixed transfection reagent and plasmid evenly to the culture dish, and gently shake back and forth and side to side to mix.

[0090] h. Place the cells back into the cell culture incubator and continue culturing for 12-60 hours. During this period, observe the cells under a fluorescence microscope to determine whether the plasmid transfection was successful.

[0091] 4. cfDNA extraction

[0092] a. Collect 1 mL of culture supernatant from cells that have been successfully transfected 18-60 h.

[0093] b. Centrifuge at 5000 rpm for 4 min to remove residual cells from the culture medium supernatant.

[0094] c. Install the vacuum pump filtration device according to the instructions of the cfDNA extraction kit (QIAGEN, QIAmp, Circulating Nucleic Acid, catalog number 55114), take the QIAamp Mini centrifuge column out of the refrigerator and equilibrate at room temperature for at least 1 hour, heat the water bath to 60°C and the metal bath to 56°C.

[0095] d. Transfer 100 μL of QIAGEN proteinase K, 1 mL of culture medium supernatant, and 0.8 mL of ACL buffer sequentially into a 50 mL centrifuge tube and vortex to mix. Ensure vortex formation in the tube; the sample and ACL buffer must be thoroughly mixed to produce a homogeneous solution to ensure effective lysis.

[0096] e. Incubate in a water bath at 60℃ (±1℃) for 30 (±1) min.

[0097] f. Add 1.8 mL of buffer ACB to the test tube, vortex to mix for 15-30 seconds, and then incubate on ice for 10 minutes.

[0098] g. Insert the QIAamp Mini centrifuge column into the VacConnector on the QIAvac 24Plus. Insert the 20mL column extender into the open QIAamp Mini centrifuge column.

[0099] h. Carefully add the lysate from step 4 into the column expander on the QIAamp Mini centrifuge column. Turn on the vacuum pump. Once all the lysate has been drawn out of the column, turn off the vacuum pump and release the pressure to 0 mbar. Carefully remove and discard the column expander.

[0100] i. Add 600 μL of buffer ACW1 to the QIAamp Mini centrifuge column. Keep the column cap open and turn on the vacuum pump. Once all the buffer ACW1 in the QIAamp Mini centrifuge column has been aspirated, turn off the vacuum pump and release the pressure to 0 mbar.

[0101] j. Add 750 μL of buffer ACW2 to the QIAamp Mini centrifuge column. Keep the column cap open and turn on the vacuum pump. Once all the buffer ACW2 in the QIAamp Mini centrifuge column has been aspirated, turn off the vacuum pump and release the pressure to 0 mbar.

[0102] k. Add 750 μL of ethanol (96–100 (v / v)%) to the QIAamp Mini centrifuge column. Keep the column cap open and turn on the vacuum pump. Once all the ethanol has been drawn out of the column, turn off the vacuum pump and release the pressure to 0 mbar.

[0103] 1. Cap the QIAamp Mini centrifuge column. Remove the column from the vacuum manifold and discard the VacConnector. Place the QIAamp Mini centrifuge column into a clean 2mL rinse tube and centrifuge at the maximum speed (2,0000g; 14,000rpm) for 3 minutes.

[0104] m. Place the QIAamp Mini centrifuge column into a new 1.5m centrifuge tube, open the cap of the centrifuge column, and incubate in a metal bath at 56°C for 10 minutes to ensure the membrane is completely dry.

[0105] n. Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 30 μL of buffer AVE to the center of the QIAamp Mini column membrane, cap it, and incubate at room temperature for 3 (±0.5) min.

[0106] Centrifuge at maximum speed (20,000g; 14,000rpm) for 1 min to elute nucleic acids, and quantify the extracted nucleic acids. The obtained nucleic acids are the cfDNA standard.

[0107] In subsequent embodiments, unless otherwise specified, the relevant steps shall follow the specific operation methods described in this general embodiment.

[0108] Example 1: Preparation of cfDNA standard using the HepG2 liver cancer cell line (total transfection plasmid: 4 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0109] a. HepG2 cells were cultured using the cell culture steps described in the general embodiment, wherein the culture medium was DMEM complete medium, and the ratio of the culture medium was 45 mL DMEM basal medium: 5 mL fetal bovine serum;

[0110] b. Plasmid construction is performed using the plasmid construction steps in the general embodiments;

[0111] c. The aforementioned HepG2 cells were transfected with plasmids using the plasmid transfection steps in the general embodiment. The two plasmids used for transfection were 2 μg each, and the culture medium mixed with the plasmids was 200 μL. The transfection reagent used was 8 μL, and the culture medium mixed with the transfection reagent was 200 μL. The cells were cultured for 72 h after transfection.

[0112] d. Using the cfDNA extraction steps in the general embodiments, extract cfDNA from the culture supernatant of successfully transfected cells to obtain cfDNA standards.

[0113] Example 2: Preparation of cfDNA standard using the HepG2 liver cancer cell line (total transfection plasmid: 6 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0114] a. HepG2 cells were cultured using the cell culture steps described in the general embodiment, wherein the culture medium was DMEM complete medium, and the ratio of the culture medium was 45 mL DMEM basal medium: 5 mL fetal bovine serum;

[0115] b. Plasmid construction is performed using the plasmid construction steps in the general embodiments;

[0116] c. The aforementioned HepG2 cells were transfected with plasmids using the plasmid transfection steps in the general embodiment. The two plasmids used for transfection were 3 μg each, and the culture medium mixed with the plasmids was 300 μL. The transfection reagent used was 12 μL, and the culture medium mixed with the transfection reagent was 300 μL. The cells were cultured for 72 h after transfection.

[0117] d. Using the cfDNA extraction steps in the general embodiments, extract cfDNA from the culture supernatant of successfully transfected cells to obtain cfDNA standards.

[0118] Example 3: Preparation of cfDNA standard using the colon cancer cell line HCT116 (total transfection plasmid: 2 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0119] a. HCT116 cells were cultured using the cell culture steps described in the general embodiment, wherein the culture medium was McCoy's 5A complete medium, and the ratio of the medium was 45 mL McCoy's 5A basal medium to 5 mL fetal bovine serum.

[0120] b. Plasmid construction is performed using the plasmid construction steps in the general embodiments;

[0121] c. The HCT116 cells were transfected with plasmids using the plasmid transfection steps in the general embodiment. The two plasmids used were 1 μg each, and the culture medium mixed with the plasmids was 150 μL. The transfection reagent used was 4 μL, and the culture medium mixed with the transfection reagent was 150 μL. The cells were cultured for 72 h after transfection.

[0122] d. Using the cfDNA extraction steps in the general embodiments, extract cfDNA from the culture supernatant of successfully transfected cells to obtain cfDNA standards.

[0123] Example 4: Preparation of cfDNA standard using the colon cancer cell line HCT116 (total transfection plasmid: 4 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0124] a. HCT116 cells were cultured using the cell culture steps described in the general embodiment, wherein the culture medium was McCoy's 5A complete medium, and the ratio was 45 mL McCoy's 5A basal medium: 5 mL fetal bovine serum;

[0125] b. Plasmid construction is performed using the plasmid construction steps in the general embodiments;

[0126] c. The HCT116 cells were transfected with plasmids using the plasmid transfection steps in the general embodiment. The two plasmids used were 2 μg each, and the culture medium mixed with the plasmids was 200 μL. The transfection reagent used was 8 μL, and the culture medium mixed with the transfection reagent was 200 μL. The cells were cultured for 72 h after transfection.

[0127] d. Using the cfDNA extraction steps in the general embodiments, extract cfDNA from the culture supernatant of successfully transfected cells to obtain cfDNA standards.

[0128] Example 5: Preparation of cfDNA standard using suspension growth cell line GM24385 (total transfection plasmid: 4 μg, wherein the mass ratio of plasmid shown in sequence 5 to plasmid shown in sequence 6 is 1:1)

[0129] a. GM24385 cells were cultured using the cell culture steps described in the general embodiment, wherein the culture medium was RPMI 1640 complete medium, and the specific ratio was 45 mL RPMI 1640 basal medium: 5 mL fetal bovine serum;

[0130] b. Plasmid construction is performed using the plasmid construction steps in the general embodiments;

[0131] c. The aforementioned GM24385 cells were transfected with plasmids using the plasmid transfection steps in the general embodiment. The two plasmids used for transfection were 2 μg each, and the culture medium mixed with the plasmids was 200 μL. The transfection reagent used was 8 μL, and the culture medium mixed with the transfection reagent was 200 μL. The cells were cultured for 72 h after transfection.

[0132] d. Using the cfDNA extraction steps in the general embodiments, extract cfDNA from the culture supernatant of successfully transfected cells to obtain cfDNA standards.

[0133] Example 6: Preparation of cfDNA standard using the HepG2 liver cancer cell line (total transfection plasmid: 1 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0134] As described in Example 1, each step was performed, but the transfected plasmids were two different plasmids, each 0.5 μg (i.e., the plasmids shown in sequence: 5 and sequence: 6), and the culture medium mixed with the plasmids was 100 μL; the transfection reagent used was 2 μL, and the culture medium mixed with the transfection reagent was 100 μL.

[0135] Example 7: Preparation of cfDNA standard using the colon cancer cell line HCT116 (total transfection plasmid: 1 μg, wherein the mass ratio of the plasmid shown in sequence 5 to the plasmid shown in sequence 6 is 1:1)

[0136] As described in Example 3, each step was performed, but the transfected plasmids were two different plasmids, each 0.5 μg (i.e., the plasmids shown in sequence: 5 and sequence: 6), and the culture medium mixed with the plasmids was 100 μL; the transfection reagent used was 2 μL, and the culture medium mixed with the transfection reagent was 100 μL.

[0137] Comparative Example 1: Control experiment for preparing cfDNA standards using the HepG2 liver cancer cell line (total transfection plasmid: 4 μg, wherein the mass ratio of the plasmid shown in sequence 1 to the plasmid shown in sequence 2 is 1:1).

[0138] As described in Example 1, each step was performed, but the transfected plasmids were two different blank plasmids, each 2 μg (i.e., the plasmids shown in Sequence: 1 and Sequence: 2), and the culture medium mixed with the plasmids was 200 μL; the transfection reagent used was 8 μL, and the culture medium mixed with the transfection reagent was 200 μL.

[0139] Comparative Example 2: Control experiment using cfDNA standards prepared from the HCT116 colon cancer cell line (total transfection plasmid: 2 μg, wherein the mass ratio of the plasmid shown in sequence 1 to the plasmid shown in sequence 2 is 1:1).

[0140] As described in Example 3, each step was performed, but the transfected plasmids were 1 μg each of two blank plasmids (i.e., the plasmids shown in sequence: 1 and sequence: 2), and the culture medium mixed with the plasmids was 150 μL; the transfection reagent used was 4 μL, and the culture medium mixed with the transfection reagent was 150 μL.

[0141] Comparative Example 3: Preparation of cfDNA Standards Using Mechanically Disrupted gDNA from the HepG2 Liver Cancer Cell Line

[0142] HepG2 cells were cultured using the cell culture steps described in the general embodiments, and then the cells were harvested and gDNA was extracted (using the TianGen TIANamp Genomic DNA Kit, catalog number DP304). The obtained gDNA was mechanically fragmented (specific fragmentation method: transfer the nucleic acid sample to the fragmentation tube provided with the instrument, prepare the fragmentation volume to 130 μL, avoiding air bubble formation; place the fragmentation tube in the designated position on the instrument (Covaris M220); turn on the instrument host, computer, and software in sequence; edit the fragmentation program, setting the fragmentation parameters to Peak Power 50.0, Duty Factor 20.0, Cycles 200, and Time 150s). The resulting product is the cfDNA standard.

[0143] Comparative Example 4: Preparation of cfDNA Standards from Mechanically Disrupted gDNA from the HCT116 Liver Cancer Cell Line

[0144] HCT116 cells were cultured using the cell culture steps described in the general examples. Cells were then harvested, and gDNA was extracted (using the same kit as Comparative Example 3). The obtained gDNA was mechanically fragmented (using the same instruments and specific fragmentation method as Comparative Example 3). The resulting product was the cfDNA standard.

[0145] Comparative Example 5: cfDNA standard was prepared using the suspension-grown cell line GM24385 (total transfection plasmid: 4 μg).

[0146] As described in Example 5, each step was performed, but the transfected plasmids were two different blank plasmids, each 2 μg (i.e., the plasmids shown in sequence: 1 and sequence: 2), and the culture medium mixed with the plasmids was 200 μL; the transfection reagent used was 8 μL, and the culture medium mixed with the transfection reagent was 200 μL.

[0147] The following comparisons will examine the above examples and comparative examples in terms of cell state and cfDNA distribution, cfDNA yield, library yield and quality, and methylation sequencing.

[0148] Test Example 1. Cell State and cfDNA Distribution

[0149] Cell state was observed using a CKX53 biological microscope (Olympus CKX53) under bright-field, red fluorescence, and blue fluorescence conditions. The distribution of cfDNA in each example and comparative example was analyzed using a fully automated nucleic acid quality control analysis system (LabChip GX Touch).

[0150] The specific details of cell state and cfDNA distribution are shown in Figure 1-10.

[0151] Figure 1A shows the cell state after transfection with the empty vector plasmid in Comparative Example 1. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 1B shows the peak pattern of the cfDNA standard prepared in Comparative Example 1. It shows that there are very obvious large fragments in the cfDNA distribution, and the main peaks 1 and 2 are relatively broad.

[0152] Figure 2A shows the cell state after transfection with the plasmid in Example 1. As shown in Figure 2A, the plasmid transfection was successful, and the corresponding protein was expressed. At the same time, the cell state was good. Figure 2B shows the peak shape of the cfDNA standard prepared in Example 1. As shown in Figure 2B, the main peak 1 of the cfDNA standard is relatively concentrated, and there are no large fragments.

[0153] Figure 3A shows the cell state after transfection with the plasmid in Example 2. As shown in Figure 3A, the plasmid transfection was successful, and the corresponding protein was expressed. At the same time, the cell state was good. Figure 3B shows the peak shape of the cfDNA standard prepared in Example 2. As shown in Figure 3B, the main peak 1 of the cfDNA standard is relatively concentrated and there are no large fragments.

[0154] Figure 4A shows the cell state after transfection with the empty vector plasmid in Comparative Example 2. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 4B shows the peak pattern of the cfDNA standard prepared in Comparative Example 2. It shows that the distribution of the cfDNA standard is characterized by a very high proportion of large fragments and a very low proportion of the main peaks 1 and 2.

[0155] Figure 5A shows the cell state after transfection with the plasmid in Example 3. As shown in Figure 5A, the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 5B shows the peak shape of the cfDNA standard prepared in Example 3. As shown in Figure 5B, compared with Comparative Example 2, the proportion of large fragments in the distribution of cfDNA standard is significantly reduced, and the proportion of main peak 1 is significantly increased.

[0156] Figure 6A shows the cell state after transfection with the plasmid in Example 4. As shown in Figure 6A, the plasmid transfection was successful, and the corresponding protein was expressed. At the same time, the cell state was good. Figure 6B shows the peak shape of the cfDNA standard prepared in Example 4. As shown in Figure 6B, compared with Comparative Example 2, the proportion of large fragments in the distribution of cfDNA standard was significantly reduced, and the proportion of the main peak 1 was significantly increased.

[0157] Figure 7A shows the cell state after transfection with the empty vector plasmid in Comparative Example 5. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 7B shows the peak pattern of the cfDNA standard prepared in Comparative Example 5. It shows that the distribution of the cfDNA standard shows that the proportion of large fragments is very high, while the proportion of the main peaks 1 and 2 is relatively low.

[0158] Figure 8A shows the cell state after transfection with the plasmid in Example 5. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 8B shows the peak pattern of the cfDNA standard prepared in Example 5. It shows that in the distribution of the cfDNA standard, compared with Comparative Example 5, the proportion of large fragments is significantly reduced, and the proportion of the main peaks 1 and 2 is significantly increased.

[0159] Figure 9A shows the cell state after transfection with the plasmid in Example 6. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 9B shows the peak pattern of the cfDNA standard prepared in Example 6. It shows that the distribution of the cfDNA standard shows that the proportion of large fragments is very high, while the proportion of the main peaks 1 and 2 is relatively low.

[0160] Figure 10A shows the cell state after transfection with the plasmid in Example 7. It can be seen that the plasmid transfection was successful, the corresponding protein was expressed, and the cell state was good. Figure 10B shows the peak pattern of the cfDNA standard prepared in Example 7. It shows that the distribution of the cfDNA standard shows that the proportion of large fragments is very high, while the proportion of the main peaks 1 and 2 is relatively low.

[0161] Test Example 2. cfDNA Yield

[0162] The cfDNA samples from each example and comparative example were quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific). The specific method was as follows: the instrument was calibrated according to the manufacturer's instructions; 1 μl of sample was added to 199 μl of sample diluent, mixed thoroughly, and centrifuged, avoiding the generation of air bubbles; the mixture was then placed at room temperature in the dark for 2 minutes; the sample mixture was then placed into the sample holder of the instrument; and the concentration was detected by clicking the test button.

[0163] The cfDNA yield results are shown in Table 1 below.

[0164] Table 1 cfDNA production

[0165] The above results indicate that the embodiments of the present invention can obtain more cfDNA in the culture medium supernatant.

[0166] Test Example 3. Library Output and Quality

[0167] The EM-seq (Enzymatic Methyl Sequencing) process was adopted. Using the same kit and manufacturer-recommended procedure, cfDNA prepared in each example and comparative example was used for library preparation. The library yield was quantified using the equipment and method described in Test Example 2, and the fragment distribution of the libraries was analyzed using the fully automated nucleic acid quality control analysis system (LabChip GX Touch) described in Test Example 1. The obtained library yields are shown in Table 2 below, and the fragment distribution of the obtained libraries is shown in Figure 11.

[0168] Table 2 Library Output

[0169] The results in Table 2 above show that, with the same amount of investment in database construction, the embodiments of the present invention can achieve a higher output of documents.

[0170] The distribution of the library fragments is shown in Figure 11. It can be seen that for the peak at 100-150bp (i.e. the peak of the dimer), the fluorescence value of the example is lower than that of the comparative example. This indicates that the dimer ratio of the cfDNA standard library in the example is lower.

[0171] Test Example 4. Targeted Methylation Sequencing

[0172] After the library preparation operations performed in each embodiment and comparative example, such as in Test Example 3, were completed, DNA fragments were captured using the SureSelect Custom Panel Probe Pool and high-throughput sequencing was performed (for details, see Alexander GE et al., Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay. PLoS One, April 14, 2023; 18(4): e0283001). The sequencing results data analysis is shown in Table 3 and Figure 12 below.

[0173] CH background methylation refers to methylation in a non-GC environment, which can be calculated using the formula: CH_M_count / (CH_M_count+CH_U_count)*100. Here, CH_M_count represents the number of H-methylated sequences on the CH sequence, and CH_U_count represents the number of H-unmethylated sequences on the CH sequence. The CH sequence refers to a sequence containing only adenine, cytosine, or thymine. The number of H-methylated sequences indicates the number of cytosine sequences where methylation has occurred to form 5-methylcytosine, and correspondingly, the number of H-unmethylated sequences indicates the number of cytosine sequences where methylation has not occurred to form 5-methylcytosine. CH background methylation is an indicator for assessing false positives in methylation. Low CH levels indicate a low level of erroneous methylation introduced during NGS library construction and sequencing, suggesting high sample quality or ease of library construction.

[0174] Table 3 CH background methylation

[0175] As shown in Table 3, the CH methylation background level of the cfDNA standard prepared in the examples was lower than that in comparative examples 3 and 4, indicating that the cfDNA standard samples prepared in the examples were of higher quality and easier to construct libraries.

[0176] As shown in Figure 12, at the same GC content (especially when the GC content is greater than 0.5), the cfDNA standard prepared in the example has a higher sequencing depth, and the GC preference of the cfDNA standard prepared in the example is better than that of comparative examples 3 and 4.

Claims

1. A method for preparing a cfDNA standard, characterized in that, Includes the following steps: a1) Construct vectors expressing DFFB and / or DNASE1L3; a2) Culture the cells to be transfected; b) Transfect cells with a vector carrying the DFFB gene and / or a vector carrying the DNASE1L3 gene and culture them. c) For successfully transfected cells, collect the culture medium supernatant and extract DNA. The obtained DNA is the cfDNA standard.

2. The preparation method according to claim 1, wherein, The vectors used to express DFFB and / or the vectors used to express DNASE1L3 are mammalian gene expression vectors.

3. The preparation method as described in any of the preceding claims, wherein, The vectors used to express DFFB and / or the vectors used to express DNASE1L3 are plasmid vectors or viral vectors.

4. The preparation method as described in any of the preceding claims, wherein, The vector used to express DFFB is the plasmid shown in sequence 1, and / or the vector used to express DNASE1L3 is the plasmid shown in sequence 2.

5. The preparation method as described in any of the preceding claims, wherein, The DFFB gene was synthesized artificially or isolated from humans, and / or the DNASE1L3 gene was synthesized artificially or isolated from humans.

6. The preparation method as described in any of the preceding claims, wherein, The DFFB gene and the plasmid shown in sequence 1 are ligated to form the plasmid shown in sequence 5, and / or the DNASE1L3 gene and the plasmid shown in sequence 2 are ligated to form the plasmid shown in sequence 6.

7. The preparation method as described in any of the preceding claims, wherein, In step a2), when the cells are cultured to the logarithmic growth phase, they are passaged.

8. The preparation method as described in any of the preceding claims, wherein, The cells are either adherent cells or suspension cells.

9. The preparation method as described in any of the preceding claims, wherein, In step a2), when passaged, the cells are cultured to a confluence of 50-80%.

10. The preparation method as described in any of the preceding claims, wherein, During the transfection process in step b), the total amount of vectors carrying the DFFB gene and / or vectors carrying the DNASE1L3 gene is 1-6 μg; preferably, during the transfection process in step b), the total amount of vectors carrying the DFFB gene and vectors carrying the DNASE1L3 gene is 1-6 μg, and the mass ratio of vectors carrying the DFFB gene to vectors carrying the DNASE1L3 gene is (1-2):(1-2).

11. The preparation method as described in any of the preceding claims, wherein, During the transfection process in step b), the vector carrying the DFFB gene and / or the vector carrying the DNASE1L3 gene are mixed with 100-300 μL of culture medium to obtain the first mixture.

12. The preparation method according to claim 11, wherein, The resulting first mixture is centrifuged at 500-1500 rpm for 15-45 seconds; preferably, it is centrifuged at 1000 rpm for 30 seconds and then allowed to stand for at least 5 minutes.

13. The preparation method as described in any of the preceding claims, wherein, In step b), the transfection reagent used is 2-12 μL.

14. The preparation method as described in any of the preceding claims, wherein, In step b), the transfection reagent is mixed with 100-300 μL of culture medium to obtain a second mixture.

15. The preparation method according to claim 14, wherein, The resulting second mixture is centrifuged at 500-1500 rpm for 15-45 seconds; preferably, it is centrifuged at 1000 rpm for 30 seconds and then allowed to stand for at least 5 minutes.

16. The preparation method according to claim 15, wherein, After thoroughly mixing the first and second mixtures, add them to the container where the cells to be transfected are cultured.

17. The preparation method as described in any of the preceding claims, wherein, After transfection in step b), the cells were cultured for another 12-60 hours.

18. The preparation method as described in any of the preceding claims, wherein, The culture medium is either a complete culture medium or a minimum required culture medium.

19. The preparation method according to claim 18, wherein, The complete culture medium is a basal culture medium containing fetal bovine serum; preferably, the proportion of fetal bovine serum is 8-12 (v / v)%.

20. The preparation method according to claim 18, wherein, The minimum required culture medium is a serum-depleted culture medium; preferably Opti-MEM. TM Serum-depleted culture medium.

21. The preparation method as described in any of the preceding claims, wherein, In step c), the success of transfection is determined based on the expression of the marker gene.

22. The preparation method as described in any of the preceding claims, wherein, In step c), at least 1 mL of the culture supernatant from the successfully transfected cells is collected.

23. The preparation method as described in any of the preceding claims, wherein, The supernatant is centrifuged to remove residual cells; preferably, centrifuged at 4000-5000 rpm for at least 4 min.

24. A cfDNA standard, which is prepared by the method of any one of claims 1-23.

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