Preparation method of cfDNA standard substance and cfDNA standard substance prepared by same

Abstract

The invention relates to the field of high-throughput sequencing, in particular to a preparation method of a cfDNA standard substance and the cfDNA standard substance prepared by the preparation method. The method comprises the following steps: 1) carrying out enzyme digestion on gDNA of cells to obtain an enzyme digestion product; and 2) performing fragment screening on the obtained enzyme-digested product by using magnetic beads, wherein in the step (1), for every one million cells, the amount of the used enzyme is 0.10-0.70 U, and after the enzyme is added, incubation is carried out for at least 5 minutes at the temperature of 30-55 DEG C; in the step (1), the used enzyme is random endonuclease. The method disclosed by the invention can be suitable for different cell lines, and the prepared cfDNA standard substance is slightly different from natural cfDNA. In addition, the inventor also finds that the method disclosed by the invention can be suitable for both fresh cells and frozen cells. The cfDNA standard substance is prepared by directly using frozen cells through the method, so that the experiment time is more flexible, and the enzyme digestion batch stability is better.

Description

Technical Field

[0001] The present invention relates to the field of high-throughput sequencing, and in particular to a method for preparing cell-free DNA (cfDNA) standards and cfDNA standards prepared thereby. Background Art

[0002] The technology of high-throughput sequencing to detect cfDNA in body fluids is currently being used more and more widely. Although this technology has good application prospects, it is still in the process of development and gradual improvement. In the process of developing and establishing the technology, a large number of samples are required as experimental materials. However, the source of real samples is relatively precious, and the cost and difficulty of obtaining them are high.

[0003] Currently, there are methods in the existing technology that use specific enzymes to perform in vitro enzymatic digestion of genomic DNA (gDNA) to obtain cfDNA standards. However, the current enzymatic digestion methods all perform the enzymatic digestion experiment immediately after the cultured living cells are harvested, which results in poor time flexibility for experimental operations. In addition, cell culture passaging increases the preparation cost and is also very likely to cause cell contamination and poor stability of enzymatic digestion batches. At the same time, the cfDNA standards produced by enzymatic digestion have a high proportion of large fragments, which are quite different from natural cfDNA, and are not conducive to subsequent high-throughput sequencing. If you want to obtain cfDNA standards with a smaller proportion of large fragments, you need to perform multiple rounds of condition optimization for different cell lines, including enzyme concentration and enzymatic digestion time, which is not conducive to production.

[0004] Therefore, there is an urgent need in this field for a preparation method of cfDNA standards with more flexible experimental time, better enzyme digestion batch stability, and applicability to different cell lines. Summary of the Invention

[0005] The present invention provides a method for preparing a cfDNA standard and a cfDNA standard prepared thereby.

[0006] In one aspect, the present invention provides a method for preparing a cfDNA standard, comprising the following steps:

[0007] 1) digesting the gDNA of the cells to obtain a digestion product; and

[0008] 2) Use magnetic beads to screen the fragments of the obtained enzyme-digested products to obtain cfDNA standards.

[0009] In one embodiment, in step 1), the enzyme used is a random endonuclease, i.e., it can randomly cleave gDNA without being restricted by specific cleavage sites, for example, Atlantis double-stranded DNA enzyme (Atlantis dsDNase) or Micrococcal Nuclease (MNase), preferably Atlantis double-stranded DNA enzyme.

[0010] In one embodiment, in step 1), the cells used are non-frozen cells. In a more specific embodiment, in step 1), the cells used are non-frozen cells cultured to the logarithmic growth phase.

[0011] In another embodiment, in step 1), the cells used are frozen cells. In a more specific embodiment, in step 1), the cells used are frozen cells cultured to the logarithmic growth phase. More specifically, the frozen cells have been stored at a temperature below -1°C (e.g., -1, -5, -10, -20, -50, -80, -100, -196°C, or a sub-range consisting of any value in these ranges) for at least 1 day (e.g., at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months or half a year, or a sub-range consisting of any value in these ranges). More specifically, the freezing solution used to freeze the frozen cells includes, but is not limited to, existing commercial cell freezing solutions, complete culture medium containing about 10 (v / v)% dimethyl sulfoxide (DMSO), or fetal bovine serum (FBS) containing about 10 (v / v)% DMSO.

[0012] In one embodiment, in step 1), the amount of enzyme used is 0.10-0.70 U, for example, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65 or 0.70 U, or a subrange consisting of any value within these ranges, preferably 0.20-0.65, more preferably 0.25-0.50 U, per about 1 million cells.

[0013] In a preferred embodiment, in step 1), 0.10-0.70 U (e.g., 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65 or 0.70 U, or a subrange consisting of any value within these ranges, preferably 0.20-0.65, more preferably 0.25-0.50 U) of Atlantis double-stranded DNA enzyme is used per approximately 1 million cells.

[0014] In one embodiment, in step 1), after adding an enzyme for enzymatic cleavage of gDNA, the mixture is incubated at a certain temperature and for a certain time, for example, at a temperature of 30-55°C (for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55°C, or a subrange consisting of any value within these ranges, preferably 30-50°C, more preferably 35-45°C), for at least 5 minutes (for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, or a subrange consisting of any value within these ranges, preferably 10-60 minutes, more preferably 20-50 minutes).

[0015] In a more specific embodiment, in step 1), after adding the enzyme for digesting gDNA, incubation is performed in a water bath or a metal bath, preferably a metal bath.

[0016] In one embodiment, step 1) further includes exposing the gDNA of the cells. More specifically, the step of exposing the gDNA of the cells is performed before adding an enzyme for digesting the gDNA. More specifically, the step of exposing the gDNA of the cells can use any existing commercial cell nuclear lysis solution and / or digestion solution.

[0017] In a specific embodiment, the cell nuclear lysis solution can contain 0.20-0.30 (wt / v)% (e.g., 0.20, 0.25, 0.30 (wt / v)%) trypsin and can have a pH value of 7.0-9.0 (e.g., 7.0, 7.5, 8.0, 8.5, 9.0).

[0018] In a specific embodiment, the digestion solution can comprise 15-25 mg / mL (e.g., 15, 20, 25 mg / mL) of proteinase K, 40-60 mM (e.g., 40, 45, 50, 55, 60 mM) Tris-HCl, and can have a pH of 6.5-8.5 (e.g., 6.5, 7.0, 7.5, 8.0, 8.5).

[0019] In one embodiment, step 1) further comprises a step of lysing the cell membrane, for example, by incubating on ice, for example, incubating for at least 1 minute, for example, at least 2 minutes, at least 5 minutes.

[0020] In one embodiment, in step 1), a step of terminating the enzymatic cleavage reaction is further included. In a more specific embodiment, terminating the enzymatic cleavage reaction can be achieved by adding a reaction-terminating solution. More specifically, the reaction-terminating solution can be any existing commercial reaction-terminating solution.

[0021] In a specific embodiment, the reaction termination solution can contain 0.40-0.60 M (e.g., 0.40, 0.45, 0.50, 0.55, 0.60 M) disodium ethylenediaminetetraacetate (EDTA-2Na) and can have a pH value of 7.0-9.0 (e.g., 7.0, 7.5, 8.0, 8.5, 9.0).

[0022] In one embodiment, step 1) further comprises a step of purifying the digested gDNA after terminating the enzyme digestion reaction. More specifically, the step of purifying the digested gDNA can use any existing commercial DNA binding solution, DNA cleaning solution and / or DNA elution solution.

[0023] In a specific embodiment, the DNA binding solution can contain 3.0-6.0 M (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 M) guanidine hydrochloride, 20-30 (v / v)% (e.g., 20, 25, 30 (v / v)%) propylene glycol and / or 0.05-0.20 M (e.g., 0.05, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20 M) ammonium acetate.

[0024] In a specific embodiment, the DNA cleaning solution comprises 50-95 (v / v)% (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 (v / v)%) ethanol.

[0025] In a specific embodiment, the DNA elution buffer comprises water, eg, distilled water, eg, double distilled water (ddH2O).

[0026] In one embodiment, in step 1), about 50 μL of enzyme digestion product is obtained for every about 1 million cells.

[0027] In one embodiment, in step 1), about 3.0-7.5 μg (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5 μg, or a subrange consisting of any values within these ranges) of digested gDNA is obtained per about 1 million cells.

[0028] In one embodiment, in step 2), magnetic beads are used for screening for at least two rounds, for example, two rounds, three rounds, or more.

[0029] In one embodiment, in step 2), the surface of the magnetic beads used has functional groups capable of adsorbing DNA, such as carboxyl, amino, hydroxyl, silanol, epoxy, p-toluenesulfonyl, methanesulfonyl, thiol, and N-hydroxysuccinimide (NHS) ester groups.

[0030] In one embodiment, in step 2), the magnetic beads used can be, for example, AMPure XP magnetic beads (e.g., Product No. A63881), magnetic beads from Suzhou Weidu Biotechnology Co., Ltd. (e.g., Product No. MD0402C09), magnetic beads from Suzhou Nanovitamin Technology Co., Ltd. (e.g., Product No. MS500-SiOH), magnetic beads from Suzhou Zhiyi Microsphere Technology Co., Ltd. (e.g., Product No. Magsil), etc.

[0031] In one embodiment, in step 2), the particle size of the magnetic beads used is 0.5-3.5 μm, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5 μm, or a sub-range consisting of any value in these ranges, preferably 0.5-3.0 μm, more preferably 0.8-1.5 μm.

[0032] In one embodiment, in step 2), the concentration of the magnetic beads used is 1.0-35.0 mg / mL, for example, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 or 35.0 mg / mL, or a sub-range consisting of any value within these ranges.

[0033] In one embodiment, in step 2), in the first round of screening, for every about 50 μL of enzymatic cleavage product, the volume of magnetic beads used is 27.5-40.0 μL, for example, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5 or 40 μL, or a sub-range consisting of any value within these ranges.

[0034] In one embodiment, in step 2), in the first round of screening, the volume of magnetic beads used for the enzymatic digestion products of gDNA from every approximately 1 million cells is 27.5-40.0 μL, for example, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5 or 40 μL, or a sub-range consisting of any values within these ranges.

[0035] In one embodiment, in step 2), in the first round of screening, the volume of magnetic beads used is 27.5-40.0 μL, for example, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5 or 40 μL, or a sub-range consisting of any value within these ranges, for every about 3.0-7.5 μg of digested gDNA.

[0036] In one embodiment, in step 2), in the first round of screening, the ratio of the volume of magnetic beads used to the volume of the enzymatic cleavage product is 0.55-0.80, for example, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, or a sub-range consisting of any values in these ranges.

[0037] In one embodiment, in step 2), in the second round of screening, for every approximately 50 μL of enzymatic cleavage product, the volume of magnetic beads used is 90-150 μL, for example, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 μL, or a sub-range consisting of any values within these ranges.

[0038] In one embodiment, in step 2), in the second round of screening, for the enzymatic digestion products of gDNA from every approximately 1 million cells, the volume of magnetic beads used is 90-150 μL, for example, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 μL, or a sub-range consisting of any values within these ranges.

[0039] In one embodiment, in step 2), in the second round of screening, the volume of magnetic beads used is 90-150 μL, for example, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 μL, or a sub-range consisting of any values within these ranges, for every approximately 3.0-7.5 μg of enzyme-digested gDNA.

[0040] In one embodiment, in step 2), in the second round of screening, the ratio of the volume of magnetic beads used to the volume of the enzymatic cleavage product is 2.0-4.0, for example, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0, or a sub-range consisting of any values in these ranges.

[0041] In one embodiment, in step 2), in the screening, for every approximately 50 μL of enzymatic cleavage product, the total volume of magnetic beads used is 117.5-190.0 μL, for example, 117.5, 120.0, 125.0, 130.0, 140.0, 150.0, 160.0, 180.0 or 190.0 μL, or a sub-range consisting of any values within these ranges.

[0042] In one embodiment, in step 2), for the enzymatic digestion products of gDNA from every approximately 1 million cells in the screening, the total volume of magnetic beads used is 117.5-190.0 μL, for example, 117.5, 120.0, 125.0, 130.0, 140.0, 150.0, 160.0, 180.0 or 190.0 μL, or a sub-range consisting of any values within these ranges.

[0043] In one embodiment, in step 2), for every approximately 3.0-7.5 μg of enzyme-digested gDNA in the screening, the total volume of magnetic beads used is 117.5-190.0 μL, for example, 117.5, 120.0, 125.0, 130.0, 140.0, 150.0, 160.0, 180.0 or 190.0 μL, or a sub-range consisting of any values within these ranges.

[0044] In one embodiment, in step 2), the ratio of the total volume of magnetic beads used in the screening to the volume of the cleavage product is 2.55-4.80, for example, 2.55, 2.60, 2.70, 2.80, 2.90, 3.00, 3.50, 4.00, 4.50 or 4.80, or a sub-range consisting of any values within these ranges.

[0045] In a preferred embodiment, the method for preparing the cfDNA standard comprises the following steps:

[0046] 1) digesting the gDNA of the cells to obtain a digestion product; and

[0047] 2) Using magnetic beads to screen the resulting enzyme-digested products to obtain cfDNA standards;

[0048] Wherein, in step 1), for every approximately 1 million cells, the amount of enzyme used is 0.10-0.70 U, and after adding the enzyme, the culture is incubated at a temperature of 30-55° C. for at least 5 minutes;

[0049] In step 1), the enzyme used is a random endonuclease.

[0050] In another aspect, the present invention provides a cfDNA standard, which is prepared by the preparation method of the cfDNA standard of the present invention.

[0051] The "cfDNA standard" described in this article is a reference material used for calibration and quality control, playing an important role in the detection and analysis of cfDNA. It is an artificially prepared cfDNA sample designed to mimic natural cfDNA. It should have a fragment distribution similar to that of natural cfDNA and can be used to assess the accuracy, precision, and sensitivity of detection methods.

[0052] The numerical values in the present invention may be approximate values after rounding.

[0053] In the present invention, “about” may include any numerical value or numerical range that deviates from the present number by 10%. For example, “about 50 μL” may refer to any numerical value or numerical range within the range of 45-55 μL.

[0054] In the present invention, the "random endonuclease" is intended to cover all enzymes that can randomly hydrolyze the internal phosphodiester bonds of nucleic acid molecules to generate oligonucleotides, for example, Atlantis double-stranded DNA enzyme (Atlantis dsDNase) and Micrococcal Nuclease (MNase).

[0055] In the present invention, unless otherwise specified, "enzyme digestion product" refers to a solution containing gDNA that has been digested. Furthermore, depending on the context, "enzyme digestion product" can refer to either a digested but unpurified product or a digested and purified product.

[0056] In the present invention, the "non-frozen cells" (or "fresh cells", which can be used interchangeably in the present invention) used in step 1) means that their direct source is "non-frozen cells"; the "frozen cells" used in step 1) means that their direct source is frozen cells.

[0057] In one embodiment of the present invention, for every approximately 1 million cells, approximately 3.0-7.5 μg of digested gDNA can be obtained, corresponding to approximately 50 μL of digestion product. Therefore, the amount of reagents (e.g., the amount of magnetic beads) in the present invention can be calculated based on this.

[0058] In the present invention, regarding the names of cell lines, those skilled in the art will understand that "GM12878" can be used interchangeably with "NA12878" and are the names of the same cell line; "NCI-H520" can be used interchangeably with "H520" and are the names of the same cell line.

[0059] The method of the present invention is applicable to different cell lines, and the resulting cfDNA standards are minimally different from native cfDNA. Furthermore, the inventors have discovered that the method of the present invention can be applied to both fresh and frozen cells. Directly using frozen cells to prepare cfDNA standards using the method of the present invention allows for more flexible experimental timelines and improved enzyme digestion batch stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The fragment distribution of the enzyme digestion products under different enzyme digestion conditions is shown.

[0061] Figure 2 The fragment distribution after screening the enzyme digestion products using different amounts of magnetic beads is shown.

[0062] Figure 3-6 Demonstrates a comparison of cfDNA standards prepared using cryopreserved cells versus fresh cells DETAILED DESCRIPTION

[0063] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples. The experimental procedures described in the following examples are all conventional procedures unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0064] General Examples

[0065] 1. Cell culture (select the culture method in 1.1 or 1.2 according to the cell type)

[0066] 1.1 Adherent cell culture

[0067] a. Before the experiment, remove the complete culture medium, trypsin, and PBS buffer from the refrigerator and preheat to 37°C.

[0068] b. Wipe the outer walls of the culture medium bottle, trypsin, and PBS buffer bottle with alcohol cotton and place them on the operating table;

[0069] c. Remove the culture dish from the cell culture incubator and observe the cell growth under an inverted microscope. Collect the cells when they are in the logarithmic growth phase.

[0070] d. Place the culture dish on the operating table and use a suction pump to remove the original culture medium;

[0071] e. Slowly add 1 mL of PBS buffer (Thermo Fisher 10010023, the same below) along the side of the culture dish and wash twice;

[0072] f. Add 1 mL of 0.25 (wt / v)% trypsin to the bottom of the culture dish, aspirate and discard, and digest in an incubator (37°C) for 2-4 minutes;

[0073] g. Add 1 mL of complete culture medium (89% (v / v) basal culture medium (RPMI 1640 medium, ATCC 30-2001; for HCT116 cells, replace with McCoy's 5a medium, ATCC 30-2007) + 10% (v / v) FBS (Gibco, Catalog No. 10091148) + 1% (v / v) antibiotics (Solarbio, Catalog No. P7630), the same below) to terminate digestion, and gently pipette the cells on the bottom surface to collect the cells in a 15 mL centrifuge tube;

[0074] h. Place the centrifuge tube in a centrifuge and centrifuge at 1000 rpm for 4 minutes;

[0075] i. Add 1 mL of complete culture medium to resuspend the cells and count the cells.

[0076] 1.2 Suspension cell culture

[0077] a. Take the PBS buffer out of the refrigerator and preheat it to 37°C before the experiment.

[0078] b. Wipe the outer wall of the PBS buffer bottle with alcohol cotton and place it on the operating table;

[0079] c. Remove the culture dish from the cell culture incubator, place it on the workbench, and collect the culture medium and cells into a 15 mL centrifuge tube.

[0080] d. Place the centrifuge tube in a centrifuge and centrifuge at 1000 rpm for 4 minutes;

[0081] i. Add 1 mL of PBS buffer to resuspend the cells and count the cells.

[0082] 2. Enzyme digestion of cell gDNA

[0083] a. Transfer 1 million cells to a 1.5 mL centrifuge tube and centrifuge (200 g, 4 min);

[0084] b. Completely discard the supernatant, add 100 μL of nuclear lysis solution (0.25 (wt / v)% trypsin, adjusted to pH 8.0 with NaHCO3) to the cell pellet, and resuspend it by gently pipetting the centrifuge tube;

[0085] c. Incubate on ice for 5 minutes to lyse cell membranes;

[0086] d. Pellet the nuclei by centrifugation (200 g, 1 min) and discard the supernatant;

[0087] e. Add 100 μL of digestion solution (20 mg / mL proteinase K, 50 mM Tris-HCl, pH 7.5) and gently invert the tube three times to wash the nuclei. Centrifuge (200 g, 1 min) and discard the supernatant. Repeat once.

[0088] f. Gently resuspend the nuclei in 100 μL of digestion buffer (same composition as above);

[0089] g. Add 0.25-0.5U of Atlantis double-stranded DNA enzyme (purchased from Zymo Research) and mix gently by pipetting;

[0090] h. Incubate in a metal bath at 42°C for 20-50 minutes;

[0091] i. Add 20 μL of reaction stop solution (0.5 M EDTA-2Na, adjust pH to 8.0 with NaOH), vortex briefly to mix, and place on ice;

[0092] j. Add 600 μL DNA binding solution (5 M guanidine hydrochloride, 25 (v / v)% propylene glycol, 0.12 M ammonium acetate) to the above centrifuge tube and mix thoroughly;

[0093] k. The mixture was loaded into a nucleic acid adsorption column, placed in a collection tube, centrifuged (12000 g, 0.5 min), and the liquid was discarded;

[0094] 1. Add 300 μL DNA cleaning solution (80 (v / v)% ethanol), centrifuge (12000g, 0.5min), and discard the liquid;

[0095] m. Centrifuge the collection tube containing the nucleic acid adsorption column (12000g, 1 min);

[0096] n. Transfer the adsorption column to a clean 1.5 mL centrifuge tube and add 51 μL of DNA elution buffer (double-distilled water) to the adsorption column. Let it stand at room temperature for at least 1 minute.

[0097] o. Centrifuge (12000 g, 0.5 min) to obtain 50 μL of purified enzyme digestion product.

[0098] 3. Use magnetic beads to screen the fragments of the purified enzyme digestion products

[0099] a. First round of screening: Add 30 μL of magnetic beads (AMPure XP magnetic beads, Catalog No. A63881; particle size 1 μm; concentration 1.4 mg / mL; beads modified with carboxyl groups) to 50 μL of the purified digestion product.

[0100] After vortex mixing, let it stand for 5 minutes;

[0101] Collect the supernatant into a new tube, taking care not to absorb the magnetic beads;

[0102] b. Second round of screening: 120 μL of magnetic beads (the same as in step a) were added to the supernatant;

[0103] After vortex mixing, let it stand for 5 minutes;

[0104] Aspirate and discard the supernatant, being careful not to absorb the magnetic beads;

[0105] c. Wash the beads with 200 μL of 80 (v / v)% ethanol, discard the ethanol, and repeat the wash;

[0106] d. Open the tube cap and dry the magnetic beads;

[0107] e. Re-dissolve with 22 μL elution buffer for 5 min;

[0108] f. Aspirate 21 μL of supernatant to obtain cfDNA standard.

[0109] 4. Whole-genome sequencing library construction

[0110] IDT WGS library construction kit (xGen TM cfDNA & FFPE DNA LibraryPrep v2 MC Kit) was used to prepare the library of the prepared cfDNA standard according to the manufacturer's recommended experimental process.

[0111] 5. Nucleic Acid Quantification Method

[0112] The prepared cfDNA standards or libraries prepared from them were quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific). 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 to avoid bubbles. The mixture was incubated at room temperature in the dark for 2 minutes. The sample mixture was then placed in the instrument's sample well and the concentration was measured by clicking the test button.

[0113] 6. Analysis of Nucleic Acid Fragment Distribution

[0114] The fragment distribution of the prepared cfDNA standards or libraries prepared therefrom was analyzed using a fully automated nucleic acid quality control analysis system (LabChip GX Touch) according to the manufacturer's recommended experimental procedures.

[0115] In the subsequent embodiments, unless otherwise specified, the relevant steps involved all adopt the specific operation methods in this general embodiment.

[0116] Example

[0117] 1. Optimization of enzyme digestion conditions

[0118] GM12878 cells (human immortalized lymphoblastoid cells), RBE cells (human hepatobiliary carcinoma cells), NCI-H520 cells (human lung squamous cell carcinoma cells), and SNU449 cells (human hepatocarcinoma cells) were subjected to the cell culture treatment described in General Example 1. After completion of the culture, the cell gDNA was digested with enzymes as described in General Example 2 (enzyme digestion conditions are shown in Table 1 below) to obtain digestion products.

[0119] Table 1 Enzyme digestion conditions for different cells

[0120]

[0121]

[0122] The obtained enzyme digestion products were analyzed according to the process of General Example 6. The results are as follows: Figure 1 shown.

[0123] As can be seen from the figure, overall, large fragments account for a higher proportion of gDNA digestion products, which differs significantly from the fragment distribution of natural cfDNA. Furthermore, the optimal digestion conditions (e.g., enzyme type, dosage, and digestion temperature) vary significantly between different cell lines.

[0124] 2. Optimization of Magnetic Bead Fragment Screening

[0125] GM12878 cells, RBE cells, NCI-H520 cells, and SNU449 cells were subjected to the cell culture treatment described in General Example 1. Following completion of the culture, the gDNA was digested with enzymes as described in General Example 2 (enzyme dosage: 0.25 U, incubation in a 42°C metal bath for 40 min). Finally, the purified digested products were subjected to fragment screening using magnetic beads according to the procedures described in General Example 3. The first round of screening used 30 μL of magnetic beads, i.e., the volume ratio of the beads to the purified digested products was 0.6 (denoted as 0.6×, the same below); the second round of screening used 120 μL of magnetic beads, i.e., the volume ratio of the beads to the purified digested products was 2.4; and a total of 150 μL of magnetic beads was used for both rounds of screening, i.e., the volume ratio of the beads to the purified digested products was 3 (denoted as 3×, the same below) (the amount of magnetic beads used can be simply denoted as 0.6× + 3×, the same below), to obtain cfDNA standards.

[0126] For comparison, the aforementioned cells were prepared and the above experimental process was performed, except that the amount of magnetic beads used was 0.7×+3×.

[0127] For comparison, GM12878 cells were prepared and subjected to the above experimental process, except that the amount of magnetic beads used was 0.5×+3×.

[0128] The cfDNA standards prepared above were analyzed according to the process of General Example 6, and the results were as follows: Figure 2 shown.

[0129] As can be seen from the figure, when 30 μL of magnetic beads were used in the first round of screening and 120 μL of magnetic beads were used in the second round of screening, the proportion of large fragments decreased significantly in all cell lines. This indicates that fragment screening under these conditions can effectively remove large fragments while retaining small fragments (for example, the first and second peaks in the figure, i.e., peaks of approximately 70 bp and approximately 170 bp, the same below), making the fragment distribution closer to that of natural cfDNA.

[0130] 3. Comparison of cfDNA Standards Prepared Using Cryopreserved Cells and Fresh Cells

[0131] 3.1 Comparison of fragment distribution characteristics

[0132] GM12878 cells, RBE cells, HCT116 cells (human colon cancer cells), and H838 cells (human non-small cell lung cancer cells) were each subjected to the cell culture treatment described in General Example 1. After culture, the cells were divided into three equal parts. The first aliquot of cells (not subjected to any freezing treatment, i.e., fresh cells) was then subjected to the enzymatic cell gDNA treatment described in General Example 2 (enzyme amount: 0.25 U, incubation in a 42°C metal bath for 40 min). Finally, the purified enzymatic cleavage products were subjected to fragment screening using magnetic beads according to the procedures described in General Example 3 (the first round of screening used 30 μL of magnetic beads, i.e., the volume ratio of the magnetic beads to the volume of the purified enzymatic cleavage products was 0.6; the second round of screening used 120 μL of magnetic beads, i.e., the volume ratio of the magnetic beads to the volume of the purified enzymatic cleavage products was 2.4). The second aliquot of cells was frozen at -80°C, and the third aliquot of cells was stored in liquid nitrogen (approximately -196°C) for 3 months, after which the same procedures as the first aliquot were performed. Thus, cfDNA standards were prepared.

[0133] Then, the prepared cfDNA standard was analyzed according to the process of General Example 6, and the results were as follows: Figure 3-6 shown.

[0134] The results showed that for the various cell lines mentioned above, storing the cells at conditions as low as -196°C for 3 months did not affect the fragment distribution of the prepared cfDNA standards, which still had a low proportion of large fragments and obvious one-peak and two-peak distributions, that is, the fragment distribution was still close to the fragment distribution of natural cfDNA.

[0135] 3.2 Comparison of cfDNA Standard Yield

[0136] The yield of the cfDNA standards prepared in Section 3.1 was measured using a Qubit4 fluorometer (Thermo Fisher Scientific). The results are shown in the table below.

[0137] Table 2 Yield of cfDNA Standards

[0138]

[0139] As can be seen from the data in the table, for each of the above cell lines, there is no significant difference in the yield of cfDNA standards made from enzymatically digested frozen cells compared to the yield of cfDNA standards made from enzymatically digested fresh cells.

[0140] Comparison of libraries constructed from cfDNA standards

[0141] The cfDNA standards prepared in Section 3.1 were used to construct a library using the method in General Example 4.

[0142] Table 3 Comparison of libraries constructed from cfDNA standards

[0143]

[0144] As can be seen from the data in the table, for the various cell lines mentioned above, freezing treatment does not significantly affect the library yield of the cfDNA standards prepared by the method of the present invention.

[0145] 3.4 Comparison of cfDNA Standard Sequencing Results

[0146] Table 4 Sequencing results - insert fragment size

[0147]

[0148]

[0149] The above sequencing results once again verified that for the various cell lines mentioned above, freezing treatment would not significantly affect the insert fragment size of the cfDNA standards prepared by the method of the present invention.

[0150] Table 5. Consistency of fragment characteristics of cfDNA standards prepared from cells treated in different ways

[0151]

[0152] In the table, BPM (Break Point Motif) refers to the 1:1 sequence between the end sequence of the DNA template and the extended form of the DNA template in the transcription direction. The statistics here are the distribution of the number of motifs with a length of 6 bp at the 5' end of the inserted fragment; EDM (End Motif) is the distribution of the number of motifs with a length of 4 bp at the 5' end of the inserted fragment.

[0153] The correlation coefficients in the table are Pearson correlation coefficients calculated based on the quantitative distribution characteristics of the motifs.

[0154] The data in the above table show that for each of the above cell lines, the fragment characteristics of cfDNA standards prepared from cells treated in different ways showed high consistency (correlation coefficients were all greater than 0.9999).

[0155] Prior to the present invention, those skilled in the art generally believed that temperature fluctuations during cell freezing and thawing would produce ice crystals, causing damage and rupture of cell membranes, which in turn could lead to the risk of damage or degradation of chromosomal structural integrity. However, as previously mentioned, the inventors unexpectedly discovered that the present method can be applied to frozen cells. Directly using frozen cells to prepare cfDNA standards using the present method allows for more flexible experimental timelines and improved enzyme digestion batch stability.

Claims

1. A method for preparing a cfDNA standard, comprising the following steps: 1) digesting the gDNA of the cells to obtain a digestion product; and 2) Using magnetic beads to screen the resulting enzyme-digested products to obtain cfDNA standards; in, In step 1), the amount of enzyme used is 0.10-0.70 U per approximately 1 million cells, and after adding the enzyme, incubate at a temperature of 30-55° C. for at least 5 minutes; In step 1), the enzyme used is a random endonuclease.

2. A method according to any preceding claim, wherein: In step 1), the enzymes used include Atlantis double-stranded DNA enzyme and / or micrococcal nuclease.

3. The method according to claim 2, wherein: In step 1), the enzyme used is Atlantis double-stranded DNA enzyme.

4. A method according to any preceding claim, wherein: In step 1), the cells used are non-frozen cells; and / or, in step 1), the cells used are frozen cells.

5. A method according to any preceding claim, wherein: In step 1), the amount of the enzyme used is 0.20-0.65 U per approximately 1 million cells.

6. The method according to claim 5, wherein: In step 1), the amount of the enzyme used is 0.25-0.50 U per approximately 1 million cells.

7. A method according to any preceding claim, wherein: In step 1), after adding the enzyme for digesting gDNA, incubate at a temperature of 30-50° C. for 10-60 minutes.

8. The method according to claim 7, wherein: In step 1), after adding the enzyme for digesting gDNA, incubate at 35-45° C. for 20-50 minutes.

9. A method according to any preceding claim, wherein: In step 1), after adding the enzyme for digesting gDNA, incubation is performed in a water bath or a metal bath.

10. A method according to any preceding claim, wherein: In step 1), for every approximately 1 million cells, approximately 50 μL of enzyme digestion product is obtained; and / or, About 3.0-7.5 μg of digested gDNA was obtained.

11. A method according to any preceding claim, wherein: In step 2), magnetic beads were used for at least two rounds of screening.

12. A method according to any preceding claim, wherein: In step 2), the surface of the magnetic beads used has functional groups capable of adsorbing DNA.

13. The method according to claim 12, wherein: In step 2), the magnetic beads used have functional groups on their surfaces selected from carboxyl, amino, hydroxyl, silanol, epoxy, p-toluenesulfonyl, methanesulfonyl, thiol and / or N-hydroxysuccinimide (NHS) ester groups.

14. A method according to any preceding claim, wherein: In step 2), the particle size of the magnetic beads used is 0.5-3.5 μm.

15. The method according to claim 14, wherein In step 2), the particle size of the magnetic beads used is 0.5-3.0 μm.

16. The method according to claim 15, wherein In step 2), the particle size of the magnetic beads used is 0.8-1.5 μm.

17. A method according to any preceding claim, wherein: In step 2), the concentration of the magnetic beads used is 1.0-35.0 mg / mL.

18. A method according to any preceding claim, wherein: In step 2), in the first round of screening, for every approximately 50 μL of enzyme digestion product, the volume of magnetic beads used is 27.5-40.0 μL; and / or, In step 2), in the first round of screening, for the enzyme digestion products of gDNA from approximately 1 million cells, the volume of magnetic beads used is 27.5-40.0 μL; and / or, In step 2), in the first round of screening, the volume of magnetic beads used was 27.5-40.0 μL for each approximately 3.0-7.5 μg of enzyme-digested gDNA.

19. A method according to any preceding claim, wherein: In step 2), in the first round of screening, the ratio of the volume of the magnetic beads used to the volume of the enzyme-digested product is 0.55-0.

80.

20. A method according to any preceding claim, wherein: In step 2), in the second round of screening, for every approximately 50 μL of enzyme digestion product, the volume of magnetic beads used is 90-150 μL; and / or, In step 2), in the second round of screening, for the enzyme digestion products of gDNA from approximately 1 million cells, the volume of magnetic beads used is 90-150 μL; and / or, In step 2), in the second round of screening, the volume of magnetic beads used was 90-150 μL for each approximately 3.0-7.5 μg of enzyme-digested gDNA.

21. The method according to any one of the preceding claims, wherein in step 2), in the second round of screening, the ratio of the volume of the magnetic beads to the volume of the enzyme-digested product is 2.0-4.

0.

22. A method according to any preceding claim, wherein: In step 2), in the screening, for every approximately 50 μL of enzyme digestion product, the total volume of magnetic beads used is 117.5-190.0 μL; and / or, In step 2), in the screening, for the enzyme digestion products of gDNA from approximately 1 million cells, the total volume of magnetic beads used is 117.5-190.0 μL; and / or, In step 2), in the screening, the total volume of magnetic beads used was 117.5-190.0 μL for each approximately 3.0-7.5 μg of enzyme-digested gDNA.

23. The method according to any one of the preceding claims, wherein in step 2), the ratio of the total volume of magnetic beads used in the screening to the volume of the enzyme cleavage product is 2.55-4.

80.

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

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