Method for preparing cfDNA-like DNA fragments

Abstract

Disclosed are cfDNA-like DNA fragments and a widely applicable method for preparing cfDNA-like DNA fragments, which produces high yield and / or large amounts of cfDNA-like fragments. The cfDNA-like DNA fragments prepared according to the method of the present disclosure accurately reflect the size distribution and diagnostic assay performance of natural cfDNA, and thus can be used for assay method validation, internal assay quality control and external assay quality evaluation, with high reproducibility and consistency.

Description

Technical Field

[0001] This invention relates to cell-free DNA (cfDNA) and methods for its preparation; particularly to detection methods for applications such as non-invasive prenatal testing (NIPT). Background Technology

[0002] Many common human diseases are linked to mutations present in the human genome, a large portion of which are heritable or may occur during fertilization or early embryonic development. Early detection of specific human conditions or diseases, such as fetal chromosomal abnormalities, is desirable, enabling clinical / therapeutic interventions that may improve patient outcomes.

[0003] Screening for disease-related genetic perturbations is a routine practice in healthcare, with the most common example being non-invasive prenatal testing (NIPT), including prenatal screening for trisomy 21, 18, and 13. However, currently available diagnostic kits for early detection of diseases and the generation of diagnoses (rather than simply screening for a condition or disease) often require invasive procedures (such as amniocentesis or chorionic villus sampling), which increases the risk of adverse reactions such as infection transmission, Rh factor sensitization, preterm birth, and miscarriage. Furthermore, these invasive procedures are expensive and technically demanding, making them unsuitable for widespread use. Therefore, there is a need to develop improved diagnostic methods for NIPT.

[0004] Current NIPT methods detect cell-free DNA (cfDNA), a small fraction of maternal blood derived from the fetus, to determine whether the fetal cfDNA component indicates one or more genetic abnormalities. cfDNA typically consists of unstable and short-lived small DNA molecules approximately 170 base pairs (bp) in length, present in low concentrations in maternal whole blood, such as 0–100 ng / mL. Furthermore, while the median fetal fraction of cfDNA is approximately 11% in the first trimester of pregnancy, it may be less than 4% in the early stages of pregnancy when NIPT is recommended (Wang et al.). Prenat. Diagn (33:662-666, 2013). Given the scarcity of cfDNA, especially fetal cfDNA, clinical samples are invaluable research materials, but their availability is limited. Therefore, one of the main bottlenecks in the development of NIPT diagnostic tests is the availability of reference cfDNA samples, as ideal reference cfDNA samples are derived from clinical samples. Therefore, there is a need to develop cfDNA-like reference samples to replace clinical samples, while maintaining similar detection performance.

[0005] Extensive research has been conducted to develop methods for producing cfDNA-like samples with performance similar to native cfDNA samples. However, the size distribution and fragmentation patterns present in native cfDNA complicate the development of cfDNA-like samples. While the exact mechanisms of cfDNA formation are not fully elucidated, the size distribution of cfDNA is very similar to that of DNA degraded during apoptosis, although cfDNA production is not necessarily dependent on apoptosis. Many methods for obtaining cfDNA-like fragments involve sonication or enzymatic digestion of longer genomic DNA to obtain short DNA fragments of similar length to cfDNA, such as approximately 150 to 200 bp. However, cfDNA-like fragments derived from sonicated or enzymatically digested DNA often have significant limitations. It has been reported that the fragmentation characteristics of cell-free DNA in plasma can differ significantly from those of genomic DNA, resulting in DNA fragments that differ from native cfDNA in sequence information and fragment length. cfDNA-like fragments produced by these methods typically perform poorly in NIPT assays, leading to erroneous conclusions and limiting their application in kit development.

[0006] Therefore, considering existing technologies, we desire more methods to generate cfDNA (or cfDNA-like fragments) with properties similar to natural cfDNA, which behave similarly to natural cfDNA in diagnostic analyses. These methods could be particularly beneficial as reference materials for NIPT detection methods, and in the development of NIPT kits, which may include dPCR or qPCR assays.

[0007] The present invention aims to overcome one or more problems found in the prior art. Summary of the Invention

[0008] This invention relates to one or more methods for preparing cfDNA; particularly for the development of detection methods such as non-invasive prenatal testing (NIPT) analysis. The methods of this invention facilitate achieving an ideal balance between the amount of cfDNA available from biological / cellular samples and the size distribution of the obtained cfDNA fragments. In particular, cfDNA samples obtained by the methods of this invention offer improved performance in mimicking naturally derived cfDNA samples compared to alternative methods for artificially obtaining cfDNA-containing materials.

[0009] In one aspect, a method for generating multiple cfDNA-like DNA fragments is provided, the method comprising: i. providing a target cell line; ii. contacting cells of the target cell line with a medium containing DMSO; and iii. incubating the cells of the target cell line with the medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments.

[0010] In another aspect, a method is provided for generating multiple cfDNA-like DNA fragments from a target adherent cell line, the method comprising: i. providing a target adherent cell line; ii. removing a culture medium from the target adherent cell line; iii. contacting cells of the target adherent cell line with a culture medium containing DMSO; and iv. incubating the cells of the target adherent cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0011] In another aspect, a method is provided for generating multiple cfDNA-like DNA fragments from a target adherent cell line, the method comprising: i. providing the target adherent cell line in a culture medium; ii. adding DMSO to the culture medium in contact with the target adherent cell line; iii. incubating the cells of the target adherent cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0012] In another aspect, a method is provided for generating multiple cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing a target suspension cell line; ii. isolating cells of the target suspension cell line from a culture medium; iii. contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO; and iv. incubating the isolated cells of the target suspension cell line with the culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0013] In another aspect, a method is provided for generating multiple cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing the target suspension cell line in a culture medium; ii. adding DMSO to the culture medium in contact with the target suspension cell line; iii. incubating isolated cells of the target suspension cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0014] In implementations of these aspects, target cell lines are incubated with a medium containing DMSO to induce apoptosis in the target cell lines and generate cfDNA-like DNA fragments, and the cfDNA-like DNA fragments are released into a medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0015] In an implementation scheme, the method may further include extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

[0016] Advantageously, according to the implementation scheme, the cfDNA-like DNA fragment has the desired length. Therefore, in the implementation scheme, the method further includes obtaining a cfDNA-like DNA fragment of the desired length. Suitablely, the ideal length of the cfDNA-like DNA fragment is between approximately 150 bp and 170 bp, for example, approximately 160 bp.

[0017] In the implementation scheme, the culture medium containing DMSO contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO.

[0018] According to the implementation plan, the target cell line cells are incubated with a medium containing DMSO for approximately 0.5 to 8 hours, approximately 0.5 to 7 hours, approximately 1 to 7 hours, approximately 1 to 6 hours, approximately 2 to 6 hours, approximately 2 to 5 hours, approximately 2.5 to 5 hours, approximately 2.5 to 4.5 hours, approximately 3 to 4.5 hours, or approximately 3 to 4 hours.

[0019] In the implementation scheme, the culture medium containing DMSO may also contain about 0.5% to 20% FBS, about 1% to 10% FBS, about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS.

[0020] According to the implementation plan, dPCR or qPCR can be performed on cfDNA-like DNA fragments to obtain the relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region. The relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region, can be determined by the first chromosome ratio of the first chromosome to the second chromosome. The first chromosome ratio of the cfDNA-like DNA fragments is advantageously the same, substantially the same, or similar to the chromosome ratio obtained from natural patient cfDNA samples.

[0021] In the implementation scheme, the first chromosome may be known or expected to be aneuploid in the target cell line, and the second chromosome may be known or expected to be euploid in the target cell line.

[0022] In the implementation, the first chromosome ratio of the cfDNA-like DNA fragment may appropriately differ from the chromosome ratio obtained from the patient's cfDNA sample by about 15%, for example, by about 12.5%, about 10%, about 7.5%, about 5%, about 2.5%, or about 1%, for example, about 10%.

[0023] In the implementation, the first chromosome ratio may be between about 1.40 to 1.60, about 1.42 to 1.58, about 1.44 to 1.56, about 1.46 to 1.54 or about 1.48 to 1.52, preferably about 1.5.

[0024] In another aspect, a method for generating multiple cfDNA-like DNA fragments is provided, the method comprising: i. providing a target cell line; ii. lysing the cells of the target cell line with a buffer containing ATAC-RSB to obtain target cell line DNA; and iii. digesting the DNA of the target cell line with a micrococcal nuclease (Mnase) to generate cfDNA-like DNA fragments.

[0025] In another aspect, methods for generating multiple cfDNA-like DNA fragments by any aspect and / or embodiment of this disclosure are provided. For example, by a method comprising the steps of: i. providing a target cell line; ii. contacting cells of the target cell line with a medium containing DMSO; and iii. incubating the cells of the target cell line with a medium containing DMSO to induce apoptosis of the target cell line and generate multiple cfDNA-like DNA fragments.

[0026] In another aspect, multiple cfDNA-like DNA fragments are provided, which are generated by the following methods: i. providing a target adherent cell line; ii. removing the culture medium from the target adherent cell line; iii. contacting the cells of the target adherent cell line with a culture medium containing DMSO; iv. incubating the cells of the target adherent cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0027] In another aspect, several cfDNA-like DNA fragments are provided, which are prepared by: i. providing a target adherent cell line in a culture medium; ii. adding DMSO to the culture medium in contact with the target adherent cell line; iii. incubating the target adherent cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0028] In another aspect, multiple cfDNA-like DNA fragments are provided, which are generated by: i. providing a target suspension cell line; ii. isolating cells of the target suspension cell line from a culture medium; iii. contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO; iv. incubating the isolated cells of the target suspension cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0029] In another aspect, multiple cfDNA-like DNA fragments are provided, which are generated by: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium in contact with the target suspension cell line; iii. incubating isolated cells of the target suspension cell line with a culture medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0030] In implementations of these aspects, target cell lines are incubated with a medium containing DMSO to induce apoptosis in the target cell lines and generate cfDNA-like DNA fragments, and the cfDNA-like DNA fragments are released into a medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0031] In the implementation scheme, the method for obtaining multiple cfDNA-like DNA fragments further includes extracting the cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

[0032] According to the implementation plan, the cfDNA-like DNA fragment has the desired length. For example, the ideal length of the cfDNA-like DNA fragment can be between approximately 150 bp and 170 bp; particularly approximately 160 bp.

[0033] In the embodiments, multiple cfDNA-like DNA fragments can be obtained by a method in which the DMSO-containing medium contains approximately 25%, approximately 24%, approximately 23%, approximately 22%, approximately 21%, approximately 20%, approximately 19%, approximately 18%, approximately 17%, approximately 16%, or approximately 15% DMSO. In the embodiments, the DMSO-containing medium may also contain approximately 0.5% to 20% FBS, approximately 1% to 10% FBS, approximately 2% to 9% FBS, approximately 3% to 8% FBS, approximately 4% to 7% FBS, or approximately 5% to 6% FBS. In the embodiments, to obtain multiple cfDNA-like DNA fragments, target cell lines can be incubated with the DMSO-containing medium for approximately 0.5 to 8 hours, approximately 0.5 to 7 hours, approximately 1 to 7 hours, approximately 1 to 6 hours, approximately 2 to 6 hours, approximately 2 to 5 hours, approximately 2.5 to 5 hours, approximately 2.5 to 4.5 hours, approximately 3 to 4.5 hours, or approximately 3 to 4 hours.

[0034] In another aspect, multiple cfDNA-like DNA fragments are provided, which are generated by the following processes: i. providing a target cell line; ii. lysing the target cell line cells with a buffer containing ATAC-RSB to obtain target cell line DNA; and iii. digesting the target cell line DNA with micrococcal nuclease to produce cfDNA-like DNA fragments.

[0035] It should be understood that, unless otherwise stated, any feature of any aspect or embodiment of the invention may be combined with any combination of features of any other aspect or embodiment of the invention, and such combination is specifically intended to fall within the scope of this disclosure. Brief description of the attached figures

[0036] The nature and advantages of the embodiments of the present invention can be better understood by referring to the following detailed description and accompanying drawings. Those skilled in the art will understand that the invention described in the drawings is readily adaptable to variations and modifications beyond those specifically described in the drawings. It should be understood that the invention includes all such variations and modifications. The scope of the invention is not limited to the specific drawings described herein, which are for illustrative purposes only.

[0037] Figure 1 Different treatment conditions induced apoptosis to varying degrees.

[0038] Cell viability results obtained by treating cells with DMSO, monensin, cisplatin, or control medium. (A) Viability of cells from the fibroblast cell line AG05397 (purchased from Coriell; "Fibroblast 397") at 4, 12, and 24 hours after treatment with DMSO, monensin, cisplatin, or control medium. The AG05397 cell line was derived from male subjects exhibiting Down syndrome. (B) Viability of B lymphocyte cell line GM24695 (purchased from Coriell; "Lymphocyte 695") at 4, 12, and 24 hours after treatment with DMSO, monensin, cisplatin, or control medium. The GM24695 cell line was derived from healthy euploid female subjects.

[0039] Figure 2 Different treatment conditions generate cfDNA-like DNA fragments, affecting the total DNA yield.

[0040] Total DNA yield results obtained by treating cells with DMSO, monensin, cisplatin, or control medium. (A) Total DNA obtained from fibroblast 397 at 4, 12, and 24 hours post-treatment in response to DMSO, monensin, cisplatin, or control medium. (B) Total DNA obtained from lymphocyte 695 at 4, 12, and 24 hours post-treatment in response to DMSO, monensin, cisplatin, or control medium.

[0041] Figure 3 The cfDNA-like DNA fragments obtained by the method according to the present invention capture the size distribution of patient cfDNA more accurately than comparative methods known in the art.

[0042] Comparison of the size distribution of cfDNA fragments obtained from patient samples with cfDNA-like DNA fragments obtained according to the method of the present invention and prior art comparative methods. (A) Electrophoresis diagram of the size distribution of cfDNA samples obtained from patients by plasma exchange or streck tubes, and electrophoresis diagrams of two cfDNA-like DNA fragment samples obtained 4 hours after DMSO treatment according to the method of the present invention. Approximately 58.2% of the DNA fragments obtained from patient samples obtained by plasma exchange showed size differences consistent with the expected size distribution of cfDNA fragments. Approximately 72.2% of the DNA fragments obtained from patient samples obtained by streck tubes showed a size distribution consistent with the expected size distribution of cfDNA fragments. "Sample 1"—Approximately 77.4% of the cfDNA-like DNA fragments obtained according to the method of the present invention showed a size distribution consistent with the expected size distribution of cfDNA fragments. "Sample 2"—Approximately 81.5% of the cfDNA-like DNA fragments obtained according to the method of the present invention showed a size distribution consistent with the expected size distribution of cfDNA fragments. (B) Electrophoretic mapping of the size distribution of cfDNA samples obtained from patient plasma bags via streck tubes, or cfDNA samples obtained 4 hours after DMSO treatment, according to the method of the present invention or the method disclosed in US11753682B2 (BT), for 472M and fibroblast 397 cells. Approximately 77.4% of the cfDNA-like DNA fragments obtained from 472M cells according to the method of the present invention exhibit a size distribution consistent with the expected size distribution of cfDNA fragments. Approximately 81.5% of the cfDNA-like DNA fragments obtained from fibroblast 397 cells according to the method of the present invention exhibit a size distribution consistent with the expected size distribution of cfDNA fragments. Approximately 93.1% of the cfDNA-like DNA fragments obtained from a patient sample (#t13) according to the method of US11753682B2 exhibit a size distribution consistent with the expected size distribution of cfDNA fragments. This sample exhibits trisomy 13 aneuploidy syndrome. Compared to the methods of the present invention and DNA fragments obtained from patient plasma bags via streck tubes, the distribution of cfDNA-like DNA fragments obtained by the method of US11753682B2 is relatively broad. Approximately 84.2% of the cfDNA-like DNA fragments obtained from a patient-derived cfDNA sample (#xo) according to the method of US11753682B2 exhibited a size distribution consistent with the expected size distribution of cfDNA fragments. This sample exhibited monosomy X (Turner syndrome) aneuploidy syndrome. Similarly, the distribution of cfDNA-like DNA fragments obtained by the method of US11753682B2 is relatively broad compared to the methods of the present invention and DNA fragments obtained from patient plasma bags via streck tubes.Approximately 59.2% of the DNA fragments obtained via streck tube from the human plasma biological control sample (#f7) of female subjects showed a size distribution consistent with the expected size distribution of cfDNA fragments. Approximately 72.2% of the DNA fragments obtained via streck tube from the human plasma biological control sample (#726M) of male subjects showed a size distribution consistent with the expected size distribution of cfDNA fragments.

[0043] Figure 4 Different processing conditions affect the size distribution of cfDNA-like DNA fragments.

[0044] Before selecting any size of cfDNA-like DNA fragments, compare the size distribution of cfDNA-like DNA fragments obtained after treatment with DMSO, monensin, or control media. The peak of fragments consistent with the expected size distribution of cfDNA fragments is indicated by arrows, approximately 172 bp. (A) Electrophoretic graphs of the size distribution of cfDNA samples obtained from fibroblast 397 cells and lymphocyte 695 cells at 4 h, 12 h, and 24 h post-treatment, according to the method of US11753682B2. At 4 h post-treatment, the cfDNA-like DNA fragments obtained from fibroblast 397 cells show a peak consistent with the expected size distribution of cfDNA, for example, approximately 172 bp. At 4 h post-treatment, the cfDNA-like DNA fragments obtained from lymphocyte 695 cells show a peak consistent with the expected size distribution of cfDNA, for example, approximately 172 bp. At 8 h and 24 h, the DNA obtained from lymphocyte 695 cells shows a distinct peak with a size distribution consistent with the expected size distribution of high molecular weight gDNA. (B) Electrophoretic images of the size distribution of cfDNA samples obtained from fibroblast 397 cells and lymphocyte 695 cells after DMSO treatment at 4 hours, 12 hours, and 24 hours post-treatment, according to the method of the present invention. At 4 hours post-treatment, the cfDNA-like DNA fragment obtained from fibroblast 397 cells showed a peak consistent with the expected size distribution of cfDNA, for example, about 172 bp. At 4 hours post-treatment, the cfDNA-like DNA fragment obtained from lymphocyte 695 cells showed a peak consistent with the expected size distribution of cfDNA, for example, about 172 bp. (C) Electrophoretic images of the size distribution of cfDNA samples obtained from fibroblast 397 cells and lymphocyte 695 cells after treatment with control medium at 4 hours, 12 hours, and 24 hours post-treatment, according to a comparative method of the present invention. At 4 hours post-treatment, the cfDNA-like DNA fragment obtained from fibroblast 397 cells showed a peak consistent with the expected size distribution of cfDNA, for example, about 172 bp. Four hours after treatment, cfDNA-like DNA fragments obtained from lymphocyte 695 cells showed peaks consistent with the expected size distribution of cfDNA, for example, approximately 172 bp. At 4, 8, and 24 hours, DNA obtained from lymphocyte 695 cells showed distinct peaks, with size distributions consistent with the expected size distribution of high molecular weight gDNA.

[0045] Figure 5 Different processing conditions generate cfDNA-like DNA fragments, affecting the detection of aneuploid and euploid chromosomes in aneuploidy detection.

[0046] Aneuploidy assay results were obtained by comparing chromosome ratios of cfDNA-like DNA fragments generated by dPCR from euploid patient control samples and aneuploid cell lines fibroblast 397 and lymphocyte 695 cells under different treatment conditions and at post-treatment time points. Each numbered panel describes the chromosome ratios of each comparative chromosome pair indicated by the X-axis marker under the following conditions: 1: Euploid patient control; 2: cfDNA-like DNA fragment from fibroblast 397 cells 4 hours after DMSO treatment; 3: cfDNA-like DNA fragment from fibroblast 397 cells 4 hours after treatment with control medium; 4: cfDNA-like DNA fragment from fibroblast 397 cells 12 hours after treatment with control medium; 5: cfDNA-like DNA fragment from fibroblast 397 cells 12 hours after monensin treatment; 6: cfDNA-like DNA fragment from fibroblast 397 cells 24 hours after monensin treatment; 7: cfDNA-like DNA fragment from fibroblast 397 cells 4 hours after monensin treatment. 8: cfDNA-like DNA fragment of lymphocytes 695 4 hours after DMSO treatment. 9: cfDNA-like DNA fragment of lymphocytes 695 12 hours after treatment with control medium. 10: cfDNA-like DNA fragment of lymphocytes 695 4 hours after treatment with control medium. 11: cfDNA-like DNA fragment of lymphocytes 695 12 hours after monensin treatment. 12: cfDNA-like DNA fragment of lymphocytes 695 4 hours after monensin treatment. Detailed Implementation

[0047] All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, such as molecular genetics, cell culture, organic chemistry, and nucleic acid chemistry and hybridization.

[0048] Generally, the nomenclature used in this article and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are well known and commonly used in the field.

[0049] Unless otherwise stated, the practice of this invention employs conventional techniques of molecular biology and genetics, cell culture, organic chemistry, and nucleic acid chemistry and hybridization, techniques within the capabilities of a person skilled in the art. Standard techniques are used for nucleic acid synthesis.

[0050] These techniques and procedures are generally performed according to conventional methods in the field (see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0051] Those skilled in the art will understand that the invention described herein is readily adaptable to variations and modifications beyond the specific description. It should be understood that the invention encompasses all such variations and modifications. The invention also includes all steps and features individually or jointly mentioned or indicated in this specification, as well as any and all combinations of any two or more of the stated steps or features. The scope of the invention is not limited to the specific embodiments described herein, which are for illustrative purposes only.

[0052] As used herein, the term "comprising" means that any one of the listed elements must be included, and other elements may be optionally included. "consistently composed of" means that any of the listed elements must be included, excluding elements that would materially affect the essential and novel features of the listed elements, and other elements may be optionally included. "composed of" means excluding all elements other than those listed. Embodiments defined by each of these terms are within the scope of this invention. It should also be understood that the term "and / or" as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0053] As used herein, the terms “a,” “an,” or “the” can include not only an aspect having one member but also a field having multiple members. For example, the singular forms “a,” “an,” and “the” may include plural references unless the context explicitly specifies otherwise. Thus, for example, a reference to “bead” can include multiple such beads, a reference to “sequence” can include a reference to one or more sequences known to those skilled in the art, and so on.

[0054] the term:

[0055] As used herein, the term "deoxyribonucleic acid base" generally comprises a nucleobase, a deoxyribose sugar, and at least one phosphate group. The nucleobase is typically a heterocyclic ring. Nucleosides include, but are not limited to, purines and pyrimidines, and more specifically include, but are not limited to, adenine, guanine, thymine, and cytosine. The sugar is typically a pentose sugar. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Phosphate may be attached to the 5' or 3' side of the nucleotide. For the purposes of this invention, the terms "nucleotide," "deoxyribonucleic acid base," and "base" are used interchangeably.

[0056] As used herein, the term "DNA fragment" is a macromolecule containing at least two or more covalently linked deoxyribonucleotide bases, wherein the 3' and 5' ends of each base are linked by a phosphodiester bond. A DNA fragment may be double-stranded or single-stranded, or may contain one or more double-stranded regions and one or more single-stranded regions. A DNA fragment may be linear or circular and may be derived from DNA in a linear or circular conformation, in a single-stranded or double-stranded conformation, or a more highly aggregated hybridization motif. DNA and DNA fragments may include any combination of any nucleotides. One or more nucleotides in any DNA fragment of this disclosure may be modified, for example, by one or more chemical modifications comprising nucleotides. Modified nucleotides may appear alone or together with other nucleotide modifications, which may be the same or different modifications, and may be continuous or discontinuous with other nucleotide modifications, whether the same or different. Modifications include, but are not limited to, modifications that provide chemical groups that incorporate additional charge, polarizability, hydrogen bonds, electrostatic interactions, linkage sites, and functionality into nucleic acid ligand bases or the entire nucleic acid ligand.

[0057] The term “cell-free DNA” or “cfDNA” refers to extracellular DNA expelled from cells, typically obtained from the noncellular portion of bodily fluids (such as blood) or cell-free biological fluids, for example, from whole blood, urine, saliva, or other biological fluids that have been processed to remove cells. Typically, cfDNA used for analysis is obtained from whole blood that has been processed to remove cells, such as plasma or serum samples. As used herein, “natural cfDNA” or “naturally occurring cfDNA” refers to cfDNA present in a biological sample, in contrast to genomic DNA fragments obtained by other methods, such as lysis or sonication.

[0058] Typically, cfDNA is approximately 150 to 200 bp in length, primarily around 166 bp, and is not bound by theory. This corresponds to the length of DNA wrapped around nucleosomes and adapters. Without further theoretical constraints, the length of cfDNA may be due to enzymatic cleavage from germline DNA in vivo, which typically results in fragments between 150 and 200 bp in length, and / or may be a product of apoptosis and cell degradation, providing a basis for cfDNA typically having a range of lengths. Therefore, cfDNA may be released from necrotic or apoptotic cells and may be associated with apoptotic bodies, nucleosomes, or other extracellular forms.

[0059] The term "cfDNA-like DNA fragment" refers to a DNA fragment produced according to the methods disclosed herein that has properties similar to natural cfDNA and achieves similar performance in assays (such as NIPT) compared to natural cfDNA (such as cfDNA from a pregnant woman or fetus).

[0060] The term "NIPT" refers to non-invasive prenatal testing, which involves determining the risk of chromosomal abnormalities (such as trisomy 21) at birth by analyzing fetal cfDNA present in a pregnant woman's sample (usually a blood sample).

[0061] As used herein, a "gene" is a segment of nucleic acid (usually DNA) that encodes the sequence information required to produce a polypeptide or ribonucleic acid gene product. It includes the regions before and after the coding regions (5'UTR and 3'UTR), as well as the insertion sequences (introns) between individual coding segments (exons). For convenience, the term also includes control sequences required for gene expression (e.g., enhancers, silencers, promoters, terminators, etc.) and the coding and / or transcriptional regions that encode the gene product; these control sequences may be adjacent to or far from the relevant coding sequences.

[0062] The term "copy number variation" (CNV) refers to the variable copy number of a specific DNA segment between different individual genomes or between different cell or tissue types within an individual genome. Variant regions have variable lengths and are typically defined as segments longer than 1000 base pairs but shorter than 5 megabases. CNVs include both extra copies of the sequence and the loss of genetic material, which can result from duplication, deletion, or other structural changes such as recombination. These regions may or may not contain one or more genes. CNVs can be detected by qPCR or dPCR, where the copy number of a specific genomic region can be reflected in the number of molecules detected or the number of partitions containing the desired oligonucleotides originating from that specific genomic region.

[0063] As used herein, the term "diagnostic assay" refers to any assay, screening, test, or method that can be used to characterize genotypes by analyzing cfDNA samples, such as aneuploidy, copy number variants, alleles, polymorphisms, splicing variants, regulatory variants, mutations, insertions / deletions, trinucleotide repeats, premature stop codons, translocations, somatic rearrangements, gene fusions, genetic alterations, or the presence of foreign or exogenous nucleotide sequences (such as proviruses). For example, a diagnostic assay can refer to any type of nucleotide sequencing and subsequent analysis, or it can include any type of nucleic acid sequencing and subsequent analysis. A diagnostic assay can be quantitative PCR (qPCR) or digital PCR (dPCR), or it can include either qPCR or dPCR. A diagnostic assay can refer to next-generation sequencing ('NGS'), or, for example, a diagnostic test can include NGS and subsequent analysis. A diagnostic assay can refer to nucleic acid hybridization, such as DNA microarray analysis. Similarly, a diagnostic assay can include nucleic acid hybridization, such as DNA microarray analysis.

[0064] As used herein, the term "polymerase chain reaction" or "PCR" refers to a method for amplifying a specific fragment or subsequence of a target double-stranded DNA in geometric progression. PCR is well known to those skilled in the art; see, for example, U.S. Patents US 4,683,195 and US 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., ed., 1990. Exemplary PCR reaction conditions typically consist of two or three cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step, a hybridization step, and a separate extension step.

[0065] Reaction characteristics (e.g., amplification rate) or reaction output (e.g., the number of detectable molecules) of a PCR reaction can be detected and measured. These PCR methods may include detection reagents or detectable tags, which can be detected using any of a variety of detection devices. Exemplary detection methods include optical detection (e.g., fluorescence or chemiluminescence) and other methods known to those skilled in the art. As a non-limiting example, fluorescent tags can be detected using a detector device equipped with a module that generates excitation light that can be absorbed by a fluorophore and a module that detects the light emitted by the fluorophore.

[0066] In embodiments, diagnostic assays may include digital PCR (dPCR) reactions, such as droplet digital PCR (ddPCR) reactions. For example, methods for performing PCR in a separated space such as droplets are described in US 2014 / 0162266, US 2014 / 0302503, and US 2015 / 0031034, the contents of which are incorporated herein by reference. In embodiments, the QX200, QX600, or QX One droplet digital PCR system (Bio-Rad) may be used. In embodiments, diagnostic assays may include qPCR reactions.

[0067] Preparation method of cfDNA-like DNA fragments

[0068] This document discloses a method for preparing cfDNA-like DNA fragments suitable for a wide range of cell types and culture applications. The disclosed method for generating cfDNA-like DNA fragments involves inducing apoptosis in target cell lines, resulting in the release of cfDNA-like DNA fragments into the surrounding culture medium, followed by extraction and size selection. The cfDNA-like DNA fragments generated according to the method of this disclosure accurately capture the size distribution and diagnostic assay performance of native cfDNA, such as NIPT assays, while advantageously producing high yields and / or large quantities of cfDNA-like DNA fragments. Furthermore, the method disclosed herein offers numerous advantages, such as ease of implementation, short production cycles, low cost, applicability to various cell types, and easy scaling up for large-scale production.

[0069] The cfDNA-like DNA fragments prepared by the methods described herein can be widely used for assay validation, internal assay quality control, and external assay quality assessment, exhibiting high reproducibility and consistency. Exemplary assays include NIPT, which may include, but is not limited to, the detection of aneuploidy, trisomy, CNV, and microdeletion. The cfDNA-like DNA fragments prepared by the methods described herein can be used in conjunction with non-invasive cfDNA samples as well as invasive samples (e.g., from amniocentesis or chorionic villus sampling (CVS)) as references in diagnostic assays. The cfDNA-like DNA fragments generated according to these methods may be suitable as patient-like controls or reference materials for in vitro screening, testing, and / or diagnosis using circulating cell-free DNA (cfDNA) as a desired biomarker, including but not limited to the detection of aneuploidy, trisomy, and microdeletion.

[0070] In an aspect of the present invention, a method for generating a plurality of cfDNA-like DNA fragments is provided, the method comprising: i. providing a target cell line; ii. contacting the cells of the target cell line with a culture medium containing DMSO; and iii. incubating the cells of the target cell line with the culture medium containing DMSO to induce apoptosis of the cells of the target cell line and generate cfDNA-like DNA fragments.

[0071] The methods and systems disclosed herein can be used with any suitable cell line obtained or extracted from any animal. Typically, the target cell line is derived from humans, but it can also be derived from another animal, particularly mammals such as mouse model organisms, or from commercially farmed animals such as horses, cattle, sheep, or pigs; or alternatively from pets such as cats or dogs.

[0072] The target cell line may be an adherent cell line, and / or a suspension cell line, or may be cultured under conditions that allow the target cell line to exhibit characteristics of either an adherent or suspension cell line. The target cell line may form two-dimensional and / or three-dimensional cell structures, and / or may be cultured in 3D culture, and / or may contain organoids. In one embodiment, the target cell line is an adherent cell line. In an alternative embodiment, the target cell line is a suspension cell line.

[0073] Those skilled in the art are aware of suitable target cell lines, including but not limited to Lymphocyte 695 (B lymphocyte line GM24695), HeLa, Jurkat, HL-60, MCF-7, Saos-2, PC3, HepG2, A549, HEK293, HEK293T, U2OS, U-87 MG, U-251 MG, U-138 MG, SH-SY5Y, GAMG, AF22, HTERT-RPE1, HTCEpi, OE19, CACO-2, HepG2, CAPAN-2, RT4, RPTEC TERT1, NTERA-2, HEK 293, HEK 293T, SuSa, PC-3, A-431, HaCaT, SK-MEL-30, WM-115, A549, HBEC3-KT, SCLC-21H, MCF7, HTERT-HME1, SiHa, SK-BR-3, T-47d, EFO-21, BEWO, AN3-CA, HeLa, HUVEC TERT2, TIME, HSkMC, BJ hTERT+, BJ, ASC TERT1, ASCdiff, U-2197, U-2 OS, RH-30, LHCN-M2, HHSteC, HBF TERT88, FHDF / TERT166, MOLT-4, HTEC / SVTERT24-B, Daudi, HDLM-2, JURKAT, U-698, U-266 / 84, U-266 / 70, RPMI-8226, REH, Karpas-707, HEL, U-937, THP-1, NB-4, K-562, HMC-1, HL-60, HAP1.

[0074] Target cell lines can be selected based on the presence of the desired condition, especially when early detection of the condition (or related disease) is required. For example, the condition might be a chromosomal abnormality in a human fetus (e.g., trisomy 21, trisomy 18). Suitable target cell lines include any cell line exhibiting the desired condition, which may include, but are not limited to: fibroblast 397 (fibroblast line AG05397; trisomy 21), Detroit 532 (trisomy 21), UWWC1-2DS3 (Down syndrome, trisomy 21), UWWC1-DS1 (Down syndrome, trisomy 21), UWWC1-DS4 (Down syndrome, trisomy 21), WC-24-02-DS-M (Down syndrome, trisomy 21), WC-24-02-DS-O (Down syndrome, trisomy 21), and WC-24-02-DS-P (Down syndrome, trisomy 21). In the implementation plan, the target cell line is lymphocyte 695 or fibroblast 397.

[0075] Exemplary culture media used in this method may include, consist of, or consist substantially of any conventional culture media suitable for culturing target cell lines, such as, but not limited to: minimum essential medium (MEM), Dulbecco modified Eagle medium (DMEM), F-12K medium, RPMI 1640 optionally containing FBS, S-MEM (serum-free medium), McCoy's 5A medium, and basal Eagle medium (BME). In embodiments, the culture medium may include fetal bovine serum (FBS).

[0076] In some aspects and implementations, apoptosis of target cell lines is induced using a medium containing dimethyl sulfoxide (DMSO), releasing cfDNA-like DNA fragments into the DMSO-containing medium. The culture system is a closed environment, typically in a petri dish or flask, where cells are surrounded by a nutrient-rich medium. It is in this medium that any nucleic acids released by the cells accumulate and / or slowly degrade further.

[0077] The experimental data disclosed in this paper show that, compared with known apoptosis-inducing treatments in the art, apoptosis induced by DMSO can result in higher yields of cfDNA-like DNA fragments and / or improved performance in NIPT aneuploidy detection.

[0078] In one embodiment, the culture medium containing DMSO contains about 40% to 1%, about 35% to 5%, about 30% to 10%, about 25% to 15%, or about 18% to 22% DMSO. In another embodiment, the culture medium containing DMSO contains about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO. In yet another embodiment, the culture medium containing DMSO contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO, for example, about 20% DMSO.

[0079] In the implementation scheme, the culture medium containing DMSO contains up to about 20% fetal bovine serum (FBS), up to about 10% FBS, up to about 9% FBS, up to about 8% FBS, up to about 7% FBS, up to about 6% FBS, up to about 5% FBS, up to about 4% FBS, up to about 3% FBS, up to about 2% FBS, up to about 1% FBS, or up to about 0.5% FBS. In the implementation scheme, the culture medium containing DMSO contains about 0.5% to 20% FBS, about 1% to 10% FBS, about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS. In the implementation scheme, the culture medium containing DMSO contains approximately 10% FBS, approximately 9% FBS, approximately 8% FBS, approximately 7% FBS, approximately 6% FBS, approximately 5% FBS, approximately 4% FBS, approximately 3% FBS, approximately 2% FBS, approximately 1% FBS, or approximately 0.5% FBS.

[0080] In the implementation scheme, the DMSO-containing culture medium contains approximately 40% DMSO and approximately 60% FBS, approximately 35% DMSO and approximately 65% ​​FBS, approximately 30% DMSO and approximately 70% FBS, approximately 25% DMSO and approximately 75% FBS, approximately 20% DMSO and approximately 80% FBS, approximately 15% DMSO and approximately 85% FBS, approximately 10% DMSO and approximately 90% FBS, approximately 5% DMSO and approximately 95% FBS, or approximately 1% DMSO and approximately 99% FBS. In the implementation scheme, the DMSO-containing culture medium comprises approximately 25% DMSO and approximately 75% FBS, approximately 24% DMSO and approximately 76% FBS, approximately 23% DMSO and approximately 77% FBS, approximately 22% DMSO and approximately 78% FBS, approximately 21% DMSO and approximately 79% FBS, approximately 20% DMSO and approximately 80% FBS, approximately 19% DMSO and approximately 81% FBS, approximately 18% DMSO and approximately 82% FBS, approximately 17% DMSO and approximately 83% FBS, approximately 16% DMSO and approximately 84% FBS, or approximately 15% DMSO and approximately 85% FBS.

[0081] In the implementation scheme, the DMSO-containing medium comprises approximately 40% DMSO and approximately 60% of a medium containing 5% FBS; approximately 35% DMSO and approximately 65% ​​of a medium containing 5% FBS; approximately 30% DMSO and approximately 70% of a medium containing 5% FBS; approximately 25% DMSO and approximately 75% of a medium containing 5% FBS; approximately 20% DMSO and approximately 80% of a medium containing 5% FBS; approximately 15% DMSO and approximately 85% of a medium containing 5% FBS; approximately 10% DMSO and approximately 90% of a medium containing 5% FBS; approximately 5% DMSO and approximately 95% of a medium containing 5% FBS; or approximately 1% DMSO and approximately 99% of a medium containing 5% FBS.

[0082] In the implementation scheme, the DMSO-containing culture medium comprises approximately 25% DMSO and approximately 75% of a culture medium containing 5% FBS; approximately 24% DMSO and approximately 76% of a culture medium containing 5% FBS; approximately 23% DMSO and approximately 77% of a culture medium containing 5% FBS; approximately 22% DMSO and approximately 78% of a culture medium containing 5% FBS; approximately 21% DMSO and approximately 79% of a culture medium containing 5% FBS; approximately 20% DMSO and approximately 80% of a culture medium containing 5% FBS; approximately 19% DMSO and approximately 81% of a culture medium containing 5% FBS; approximately 18% DMSO and approximately 82% of a culture medium containing 5% FBS; approximately 17% DMSO and approximately 83% of a culture medium containing 5% FBS; approximately 16% DMSO and approximately 84% of a culture medium containing 5% FBS; or approximately 15% DMSO and approximately 85% of a culture medium containing 5% FBS.

[0083] In the implementation scheme, target cell lines are incubated with a medium containing DMSO to induce apoptosis. The experimental data disclosed herein indicate that the duration of incubation affects the viability of the target cell lines, and consequently appears to affect the quality of cfDNA-like DNA fragments; for example, longer incubation times produce cfDNA fragments with shorter than desired fragment length distributions, while shorter incubation times may reduce the yield of cfDNA-like DNA fragments. One advantage of the disclosed method is the achievement of a specific range of apoptosis induction, which is not bound by any particular theory and appears to improve the yield of cfDNA-like DNA fragments and NIPT detection performance, making it closer to native cfDNA.

[0084] In one embodiment, the target cell line is incubated with a DMSO-containing medium for up to about 24 hours, up to about 12 hours, up to about 6 hours, or up to about 4 hours. In another embodiment, the target cell line is incubated with a DMSO-containing medium for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours. In yet another embodiment, the target cell line is incubated with a DMSO-containing medium for about 0.5, about 1, about 2, about 3, or about 4 hours. In yet another embodiment, the target cell line is incubated with a DMSO-containing medium for about 0.5 to about 12 hours, about 1 to about 6 hours, about 2 to about 4 hours, or about 3 to about 4 hours. In yet another embodiment, the target cell line is incubated with a DMSO-containing medium at a temperature of about 37°C.

[0085] In an aspect of the invention, a method is provided for generating multiple cfDNA-like DNA fragments from a target adherent cell line, the method comprising: i. removing culture medium from the target adherent cell line; ii. contacting the cells of the target adherent cell line with a culture medium containing DMSO; iii. incubating the cells of the target adherent cell line with the DMSO-containing culture medium to induce apoptosis and generate cfDNA-like DNA fragments. Optionally, the method may include first providing a target adherent cell line.

[0086] In an aspect of the invention, a method is provided for generating multiple cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. isolating cells of the target suspension cell line from a culture medium; ii. contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO; iii. incubating the isolated cells of the target suspension cell line with the DMSO-containing culture medium to induce apoptosis and generate cfDNA-like DNA fragments. Optionally, the method may include first providing a target adherent cell line.

[0087] In one embodiment, separating cells from a target suspension cell line from a culture medium includes: separating the culture medium containing the target suspension cell line by precipitation to obtain a culture medium supernatant and precipitated target suspension cells; and removing the culture medium supernatant from the precipitated target suspension cell line. In another embodiment, the precipitated target suspension cell line can be produced by centrifugation or gravity separation. In yet another embodiment, separating cells from a target suspension cell line from a culture medium may include removing the culture medium supernatant from the precipitated target suspension cell line by aspiration.

[0088] In one embodiment, contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO includes resuspending the precipitated cells of the target suspension cell line in a culture medium containing DMSO.

[0089] In the implementation scheme, the target cell line can be obtained by a cell sorting method, which includes contacting the sorted cells with a culture medium containing DMSO, for example, sorting the target cell line into a culture medium containing DMSO.

[0090] Suitable cell sorting methods include, but are not limited to: fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), immunomagnetic cell separation, density gradient centrifugation, immunodensity cell separation, microfluidic cell sorting, buoyancy activated cell sorting, aptamer-based cell separation, complement depletion, limiting dilution or serial dilution, and micromanipulation or manual cell sorting.

[0091] In the implementation scheme, the target cell line is incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments. The cfDNA-like DNA fragments are then released into the medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0092] In an embodiment, the method further includes extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

[0093] In the implementation scheme, after incubating the target cell line with a medium containing DMSO, cfDNA-like DNA fragments can be extracted from the medium containing DMSO.

[0094] In one embodiment, extracting cfDNA-like DNA fragments from a DMSO-containing medium includes collecting a medium containing multiple cfDNA-like DNA fragments and performing DNA extraction on the medium containing multiple cfDNA-like DNA fragments to obtain a sample containing multiple cfDNA-like DNA fragments.

[0095] In one embodiment, extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments includes: collecting the culture medium containing DMSO and cfDNA-like DNA fragments; and performing DNA extraction on the culture medium containing DMSO and cfDNA-like DNA fragments to obtain a sample containing multiple cfDNA-like DNA fragments.

[0096] Various methods for extracting cfDNA from liquid samples (such as culture media) are known in the art. General methods for DNA preparation can be followed (e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001). cfDNA extraction from liquid samples can be achieved using columns, beads, magnetic beads, or other separation procedures. cfDNA can also be obtained from samples using commercially available kits for extracting cfDNA from samples, such as the QIAamp Circulating Nucleic Acid Kit (Qiagen), the QiaAmp DNA Mini Kit (Qiagen), or the Apostle Extraction kit (Beckman). In embodiments, the DNA extraction method includes using the QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0097] In the implementation plan, after DNA extraction, the proportion of cfDNA in the extracted DNA can be at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99.9%.

[0098] One advantage of this invention is that DNA does not require extensive purification, such as removal from exosomes, and thus one can benefit from one or more of the increased speed, cost, and ease of use.

[0099] In this embodiment, the cfDNA-like DNA fragment has the desired length. In this embodiment, the method includes obtaining a cfDNA-like DNA fragment of the desired length. In this embodiment, the method further includes obtaining a cfDNA-like DNA fragment of the desired length.

[0100] cfDNA-like DNA fragments can be processed by generating a cfDNA-like DNA fragment distribution with a specific fragment length distribution or a cfDNA-like DNA fragment distribution with a desired length.

[0101] In the implementation, obtaining cfDNA-like DNA fragments of the desired length includes size selection of cfDNA-like DNA fragments from a sample containing multiple cfDNA-like DNA fragments.

[0102] In an implementation scheme, obtaining a cfDNA-like DNA fragment of the desired length may include size selection of the cfDNA-like DNA fragment.

[0103] Methods for DNA size selection are well known in the art and include various techniques for separating DNA fragments with a desired length distribution. Methods employed in the art include, but are not limited to: gel electrophoretic separation of DNA fragments; cutting and purifying the cut electrophoretic gel based on a target size distribution; restriction enzyme digestion to cleave DNA at specific recognition sites to produce DNA fragments of predetermined sizes; size exclusion chromatography; and magnetic bead-based size selection methods.

[0104] DNA size selection methods typically employ magnetic or paramagnetic, including superparamagnetic or glass-coated beads, such as polymer-coated beads, to selectively bind and separate DNA fragments of a specific size range, depending on the concentration of the beads and the ratio of DNA used in the reaction to the beads.

[0105] Commonly used magnetic beads include, but are not limited to: silica-coated magnetic beads, carboxyl-modified magnetic beads, amino-blocked magnetic beads, oligonucleotide (dT)-coated magnetic beads, streptavidin-coated magnetic beads, streptavidin-blocked magnetic beads, magnetic agarose beads, and glass emulsion magnetic beads. Methods for preparing beads suitable for DNA size selection are known in the art (see: https: / / www.protocols.io / view / home-brew-spri-beads-eq2ly3mkmgx9 / v1) or, commercially available beads for DNA size selection, such as SPRIselect (Beckman) or AMPure XP beads (Beckman).

[0106] After incubating a DNA sample with magnetic beads for DNA size selection, a magnetic scaffold can be used to capture the DNA-bound beads, allowing the removal and discarding of supernatant containing unwanted DNA fragments, or obtaining supernatant containing the desired DNA fragments, optionally for subsequent size selection steps, such as bifacial size selection including additional bead incubation. A subsequent washing step further purifies the DNA-bound beads, ensuring that only the desired fragment size is retained. The purified, retained DNA fragments can then be eluted from the beads. In this embodiment, size selection of cfDNA-like DNA fragments includes the use of AMPure XPBeads (Beckman).

[0107] In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is between about 150 bp and 170 bp; optionally about 160 bp. In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is between about 50 bp and 300 bp, between about 100 bp and 250 bp, or between about 150 bp and 200 bp.

[0108] In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 bp. In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 bp. In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is approximately 150, 155, 160, 165, or 170 bp. In the implementation plan, the ideal length of the cfDNA-like DNA fragment is less than approximately 500, 400, 300, 200, or 170 bp.

[0109] One advantage of this invention is that the cfDNA-like DNA fragments generated by the disclosed method accurately capture the characteristics of native cfDNA. Experimental data disclosed herein demonstrate that, compared to methods currently employed in the art, including those involving apoptosis induction (e.g., see...),... Figure 3 The cfDNA-like DNA fragments produced by the disclosed method exhibit an ideally narrower size distribution. Furthermore, as shown in the electrophoresis data submitted herein, the DNA fragment size of each peak and the percentage of each peak of cfDNA in the sample representing the extracted and selected size closely match the fragment size of native cfDNA.

[0110] The inventors have discovered for the first time that treatment of target cell lines with DMSO (appropriately, a specific concentration of DMSO) produces DNA fragments that are improved copies of cfDNA obtained from patients, compared to methods in the art (such as US11753682B2), which can be at least demonstrated by their size distribution and performance in diagnostic assays such as aneuploidy assays, which closely approximate / "mimic" cfDNA obtained from patient samples.

[0111] In particular, the inventors have demonstrated for the first time that using DMSO as an apoptosis-inducing chemical produces cfDNA-like DNA fragments with the desired properties of cfDNA obtained from patient samples, compared to other well-known methods or chemicals used to induce apoptosis, such as monensin and cisplatin.

[0112] Furthermore, the inventors have demonstrated for the first time that the incubation time of DMSO (a culture medium appropriately containing DMSO) with the target cell line can significantly affect the properties of cfDNA-like DNA fragments and their performance in diagnostic tests such as aneuploidy detection.

[0113] It is worth noting that, compared with methods known in the art and other treatments for inducing apoptosis, the cfDNA-like DNA fragments generated by the method of the present invention have improved performance consistency in aneuploidy detection, for example, correctly detecting aneuploid chromosomes and euploid staining in all tested cell lines, and may even generate DNA fragments with the desired size distribution, but their performance is not as good as that of the cfDNA-like DNA fragments generated by the method of the present invention.

[0114] Therefore, in the embodiments, dPCR or qPCR is performed on the cfDNA-like DNA fragments obtained according to the method of the present invention to obtain the relative abundance of the first chromosome relative to the second chromosome.

[0115] In the implementation scheme, dPCR or qPCR is performed on the cfDNA-like DNA fragments obtained by the method according to the present invention to obtain the relative abundance of the first genomic region relative to the second genomic region, for example, to obtain the copy number of the first genomic region and the copy number of the second genomic region.

[0116] In the implementation scheme, the relative abundance of the first chromosome relative to the second chromosome includes the first chromosome ratio of the first chromosome to the second chromosome.

[0117] In the implementation scheme, the relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region, includes a first ratio between the first chromosome and the second chromosome or between the first genomic region and the second genomic region; wherein the relative abundance of the first chromosome relative to the second chromosome includes the first chromosome ratio.

[0118] In the implementation scheme, the first chromosome is known or expected to be aneuploid in the target cell line, and the second chromosome is known or expected to be euploid in the target cell line.

[0119] In the implementation scheme, the first chromosome is known or expected to be triploid in the target cell line, and the second chromosome is known or expected to be diploid in the target cell line; wherein the target cell line is a target adherent cell line or a target suspension cell line.

[0120] In the implementation scheme, the first chromosome ratio is between about 1.40 to 1.60, about 1.42 to 1.58, about 1.44 to 1.56, about 1.46 to 1.54, or about 1.48 to 1.52, for example, about 1.5.

[0121] In the implementation scheme, the first chromosome is known or expected to be euploid in the target cell line, and the second chromosome is known or expected to be euploid in the target cell line, wherein the target cell line is suitably a target adherent cell line or a target suspension cell line.

[0122] In the implementation scheme, the first chromosome is known or expected to be diploid in the target cell line, and the second chromosome is known or expected to be diploid in the target cell line.

[0123] In the implementation scheme, the first chromosome ratio is between about 0.9 and 1.1, between about 0.92 and 1.08, between about 0.94 and 1.06, between about 0.96 and 1.04, between about 0.98 and 1.02, for example, about 1.0.

[0124] In the implementation scheme, the first chromosome ratio of the cfDNA-like DNA fragments is similar to the chromosome ratio obtained from a patient's cfDNA sample.

[0125] In an implementation scheme, the first ratio of cfDNA-like DNA fragments may be similar to the ratio obtained from a patient's cfDNA sample; wherein the first chromosome ratio of the cfDNA-like DNA fragments is similar to the chromosome ratio obtained from a patient's cfDNA sample.

[0126] In the implementation scheme, the first chromosome ratio of the cfDNA-like DNA fragment may differ from the chromosome ratio obtained from the patient's cfDNA sample by up to about 20%, for example, by up to about 15%, up to about 12.5%, up to about 10%, up to about 7.5%, up to about 5%, up to about 2.5%, or up to about 1%, for example, between about -10% and +10%, between about -8% and +8%, between about -6% and +6%, between about -4% and +4%, or between about -2% and +2%.

[0127] In the implementation, the first ratio of cfDNA-like DNA fragments differs from the ratio obtained from the patient's cfDNA sample by up to about 20%, for example, up to about 15%, up to about 12.5%, up to about 10%, up to about 7.5%, up to about 5%, up to about 2.5%, or up to about 1%, and differs from the chromosome ratio from the patient's cfDNA sample by, for example, between about -10% and +10%, between about -8% and +8%, between about -6% and +6%, between about -4% and +4%, or between about -2% and +2%; wherein the first ratio is the first chromosome ratio.

[0128] Therefore, the cfDNA-like DNA fragments produced by the method according to the present invention may be particularly useful as a reference material for NIPT assays and for the development of NIPT assays, which may include dPCR or qPCR assays.

[0129] The method disclosed herein advantageously achieves an optimal balance between high cfDNA-like DNA fragment yield and cfDNA-like DNA fragments with NIPT assay performance similar to native cfDNA. This appears to be achieved through finely tuned apoptosis induction ranges, without being bound by any particular theory. This freedom from theoretical constraints, and the belief that specific induction of apoptosis is another benefit of this invention, is because many types of cell death exist, with apoptosis being only one pathway. Indeed, as Figure 1 , 2 As shown in Figures 5 and 6, inducing cell death alone does not appear to be sufficient to achieve the desired cfDNA-like DNA fragments, where the increase in cell death is not associated with an increase in the yield of cfDNA-like DNA fragments or the desired characteristics.

[0130] Provision of cfDNA-like DNA fragments

[0131] This article discloses cfDNA-like DNA fragments applicable to a wide range of cell types and culture applications. The disclosed cfDNA-like DNA fragments are obtained by inducing apoptosis in target cell lines, resulting in the release of the cfDNA-like DNA fragments into the surrounding culture medium, where they can then be extracted and size-selected. The provided cfDNA-like DNA fragments accurately capture the size distribution and diagnostic assay performance of native cfDNA, such as NIPT assays, and can be produced in high yields and / or large quantities. The production of the disclosed cfDNA-like DNA fragments is advantageously easy, with short production cycles, low cost, applicability to various cell types, and can be easily scaled up for large-scale production.

[0132] The cfDNA-like DNA fragments described herein can be widely used for detection method validation, internal detection quality control, and external detection quality assessment, exhibiting high reproducibility and consistency. Exemplary detections include NIPT, which may include, but is not limited to, the detection of aneuploidy, trisomy, CNV, and microdeletions. The cfDNA-like DNA fragments described herein can be used as a reference for diagnostic analysis in conjunction with non-invasive cfDNA samples and invasive samples; for example, samples derived from amniocentesis or CVS. The cfDNA-like DNA fragments described herein may be suitable as patient-like controls or reference materials for in vitro screening, testing, and / or diagnosis using circulating cell-free DNA (cfDNA) as a desired biomarker, including but not limited to the detection of aneuploidy, trisomy, and microdeletions.

[0133] Typically, the method of the present invention includes the following steps: contacting a plurality of target cells with a culture medium containing DMSO, and incubating the cells with the DMSO-containing culture medium to induce apoptosis; thereby generating a plurality of cfDNA-like DNA fragments. These cells may be derived from a target cell line. The method of the present invention may include providing a target cell line and contacting its cells with a culture medium containing DMSO.

[0134] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0135] i. Provide target cell lines;

[0136] ii. Contact the target cell line with a culture medium containing DMSO; and

[0137] iii. Incubate the target cell line with a medium containing DMSO to induce apoptosis in the target cell line and generate multiple cfDNA-like DNA fragments.

[0138] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0139] i. Provide target cell lines

[0140] ii. Lyse the target cell line cells with a buffer containing ATAC-RSB to obtain target cell line DNA; and

[0141] iii. Digest the DNA of the target cell line with micrococcal nuclease to produce cfDNA-like DNA fragments.

[0142] In the implementation plan, the target cell line is an adherent cell line.

[0143] In the implementation plan, the target cell line is a suspension cell line.

[0144] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0145] i. Provide targeted adherent cell lines;

[0146] ii. Remove the culture medium from the target adherent cell line;

[0147] iii. Contact the target adherent cell line with a culture medium containing DMSO;

[0148] iv. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0149] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0150] i. Provide target adherent cell lines in the culture medium;

[0151] ii. Add DMSO to the culture medium in contact with the target adherent cell line;

[0152] iii. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0153] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0154] i. Provide target suspension cell lines;

[0155] ii. Isolate cells from the target suspension cell line from the culture medium;

[0156] iii. Contact the isolated cells of the target suspension cell line with a culture medium containing DMSO;

[0157] iv. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0158] In this invention, a plurality of cfDNA-like DNA fragments are provided, which are generated by the following method:

[0159] i. Provide the target suspension cell line in the culture medium;

[0160] ii. Add DMSO to the culture medium in contact with the target suspension cell line;

[0161] iii. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0162] In the implementation scheme, isolating cells of the target suspension cell line from the culture medium includes:

[0163] The culture medium containing the target suspended cell line is separated by precipitation to obtain a culture supernatant and a precipitated target suspended cell line; and

[0164] Remove the culture medium supernatant from the precipitated target suspension cell line.

[0165] In the implementation plan, the precipitated target suspension cell lines are generated by centrifugation or gravity separation.

[0166] In one implementation, separating the target suspension cell line from the culture medium includes removing the culture medium supernatant from the precipitated target suspension cell line by aspiration.

[0167] In one embodiment, contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO includes resuspending the precipitated cells of the target suspension cell line in a culture medium containing DMSO.

[0168] In the implementation scheme, the target cell line is incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments. The cfDNA-like DNA fragments are then released into the medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0169] In the implementation scheme, the method further includes extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

[0170] In the implementation scheme, the extraction of cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments includes:

[0171] Collect culture media containing DMSO and cfDNA-like DNA fragments; and

[0172] DNA extraction was performed on a culture medium containing DMSO and cfDNA-like DNA fragments to obtain a sample containing multiple cfDNA-like DNA fragments.

[0173] In the implementation scheme, the cfDNA-like DNA fragment has the required length.

[0174] In an implementation, the method further includes size selection of cfDNA-like DNA fragments in a sample containing multiple cfDNA-like DNA fragments to obtain cfDNA-like DNA fragments of desired length.

[0175] In the implementation scheme, size selection includes single-core body size selection.

[0176] In the implementation plan, the ideal length of the cfDNA-like DNA fragment is between approximately 50 bp and 300 bp, between approximately 100 bp and 250 bp, or between approximately 150 bp and 200 bp.

[0177] In the implementation scheme, the ideal length of the cfDNA-like DNA fragment is between approximately 150 bp and 170 bp; optionally approximately 160 bp.

[0178] In the implementation scheme, the culture medium containing DMSO contains about 40% to 1%, about 35% to 5%, about 30% to 10%, or about 25% to 15% DMSO.

[0179] In the implementation scheme, the culture medium containing DMSO contains about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO.

[0180] In the implementation scheme, the culture medium containing DMSO contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO.

[0181] In the implementation scheme, the culture medium containing DMSO contains up to about 20% fetal bovine serum (FBS), up to about 10% FBS, up to about 9% FBS, up to about 8% FBS, up to about 7% FBS, up to about 6% FBS, up to about 5% FBS, up to about 4% FBS, up to about 3% FBS, up to about 2% FBS, up to about 1% FBS, or up to about 0.5% FBS.

[0182] In the implementation scheme, the culture medium containing DMSO contains about 0.5% to 20% FBS, about 1% to 10% FBS, about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS.

[0183] In the implementation scheme, the culture medium containing DMSO contains approximately 10% FBS, approximately 9% FBS, approximately 8% FBS, approximately 7% FBS, approximately 6% FBS, approximately 5% FBS, approximately 4% FBS, approximately 3% FBS, approximately 2% FBS, approximately 1% FBS, or approximately 0.5% FBS.

[0184] In the implementation scheme, the DMSO-containing culture medium comprises approximately 40% DMSO and approximately 60% containing 5% FBS, approximately 35% DMSO and approximately 65% ​​containing 5% FBS, approximately 30% DMSO and approximately 70% containing 5% FBS, approximately 25% DMSO and approximately 75% containing 5% FBS, approximately 20% DMSO and approximately 80% containing 5% FBS, approximately 15% DMSO and approximately 85% containing 5% FBS, approximately 10% DMSO and approximately 90% containing 5% FBS, approximately 5% DMSO and approximately 95% containing 5% FBS, or approximately 1% DMSO and approximately 99% containing 5% FBS.

[0185] In the implementation scheme, the DMSO-containing culture medium comprises approximately 25% DMSO and approximately 75% containing 5% FBS, approximately 24% DMSO and approximately 76% containing 5% FBS, approximately 23% DMSO and approximately 77% containing 5% FBS, approximately 22% DMSO and approximately 78% containing 5% FBS, approximately 21% DMSO and approximately 79% containing 5% FBS, approximately 20% DMSO and approximately 80% containing 5% FBS, approximately 19% DMSO and approximately 81% containing 5% FBS, approximately 18% DMSO and approximately 82% containing 5% FBS, approximately 17% DMSO and approximately 83% containing 5% FBS, approximately 16% DMSO and approximately 84% containing 5% FBS, or approximately 15% DMSO and approximately 85% containing 5% FBS.

[0186] In the implementation scheme, the DMSO-containing culture medium comprises approximately 40% DMSO and approximately 60% containing 5% FBS, approximately 35% DMSO and approximately 65% ​​containing 5% FBS, approximately 30% DMSO and approximately 70% containing 5% FBS, approximately 25% DMSO and approximately 75% containing 5% FBS, approximately 20% DMSO and approximately 80% containing 5% FBS, approximately 15% DMSO and approximately 85% containing 5% FBS, approximately 10% DMSO and approximately 90% containing 5% FBS, approximately 5% DMSO and approximately 95% containing 5% FBS, or approximately 1% DMSO and approximately 99% containing 5% FBS.

[0187] In the implementation scheme, the DMSO-containing culture medium comprises approximately 25% DMSO and approximately 75% containing 5% FBS, approximately 24% DMSO and approximately 76% containing 5% FBS, approximately 23% DMSO and approximately 77% containing 5% FBS, approximately 22% DMSO and approximately 78% containing 5% FBS, approximately 21% DMSO and approximately 79% containing 5% FBS, approximately 20% DMSO and approximately 80% containing 5% FBS, approximately 19% DMSO and approximately 81% containing 5% FBS, approximately 18% DMSO and approximately 82% containing 5% FBS, approximately 17% DMSO and approximately 83% containing 5% FBS, approximately 16% DMSO and approximately 84% containing 5% FBS, or approximately 15% DMSO and approximately 85% containing 5% FBS.

[0188] In the implementation plan, the target cell line cells are incubated with a medium containing DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours, or up to about 4 hours.

[0189] In the implementation plan, the target cell line cells are incubated with a culture medium containing DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

[0190] In the implementation plan, the target cell line cells are incubated with a medium containing DMSO for about 0.5, about 1, about 2, about 3, or about 4 hours.

[0191] In the implementation scheme, the target cell line is incubated with a medium containing DMSO at a temperature of approximately 37°C.

[0192] The implementation plan includes determining copy number variability of DNA regions in target cell lines based on the presence of cfDNA-like DNA fragments.

[0193] The implementation plan includes performing dPCR or qPCR reactions.

[0194] In the implementation plan, dPCR or qPCR is performed on cfDNA-like DNA fragments to determine the fetal fraction and / or copy number variability of DNA regions in the biological sample.

[0195] In the implementation scheme, dPCR or qPCR is performed on cfDNA-like DNA fragments to obtain the relative abundance of the first chromosome relative to the second chromosome.

[0196] In the implementation scheme, the relative abundance of the first chromosome relative to the second chromosome includes the first chromosome ratio of the first chromosome to the second chromosome.

[0197] In the implementation scheme, the relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region, includes a first ratio of the first chromosome to the second chromosome or the first genomic region to the second genomic region, wherein suitably, the relative abundance of the first chromosome relative to the second chromosome includes a first chromosome ratio.

[0198] In the implementation scheme, the first chromosome is known or expected to be aneuploid in the target cell line, and the second chromosome is known or expected to be euploid in the target cell line.

[0199] In the implementation scheme, the first chromosome is known or expected to be triploid in the target cell line, and the second chromosome is known or expected to be diploid in the target cell line, wherein the target cell line is a target adherent cell line or a target suspension cell line.

[0200] In the implementation scheme, the first chromosome ratio is between about 1.40 to 1.60, about 1.42 to 1.58, about 1.44 to 1.56, about 1.46 to 1.54, or about 1.48 to 1.52, for example, about 1.5.

[0201] In the implementation scheme, the first chromosome is known or expected to be euploid in the target cell line, and the second chromosome is known or expected to be euploid in the target cell line, wherein the target cell line is a target adherent cell line or a target suspension cell line.

[0202] In the implementation scheme, the first chromosome is known or expected to be diploid in the target cell line, and the second chromosome is known or expected to be diploid in the target cell line.

[0203] In the implementation scheme, the first chromosome ratio is between about 0.9 and 1.1, between about 0.92 and 1.08, between about 0.94 and 1.06, between about 0.96 and 1.04, between about 0.98 and 1.02, for example, about 1.0.

[0204] In the implementation plan, the first chromosome ratio of the cfDNA-like DNA fragment is the same, substantially the same, or similar to the chromosome ratio obtained from the patient's cfDNA sample.

[0205] In the implementation scheme, the first ratio of cfDNA-like DNA fragments is the same, substantially the same, or similar to the ratio obtained from the patient's cfDNA sample, wherein the first chromosome ratio of cfDNA-like DNA fragments is the same, substantially the same, or similar to the chromosome ratio obtained from the patient's cfDNA sample.

[0206] In the implementation scheme, the first chromosome ratio of the cfDNA-like DNA fragment differs from the chromosome ratio obtained from the patient's cfDNA sample by about 25%, for example, by about 20%, about 15%, about 12.5%, about 10%, about 7.5%, about 5%, about 2.5%, or about 1% compared to the chromosome ratio obtained from the patient's cfDNA sample (e.g., 10%).

[0207] In the implementation scheme, the first ratio of cfDNA-like DNA fragments differs from the ratio obtained from the patient's cfDNA sample by up to about 20%, for example, up to about 15%, up to about 12.5%, up to about 10%, up to about 7.5%, up to about 5%, up to about 2.5%, or up to about 1%, and differs from the chromosome ratio of the patient's cfDNA sample by, for example, between about -10% and +10%, between about -8% and +8%, between about -6% and +6%, between about -4% and +4%, or between about -2% and +2%. Suitably, the first ratio is the first chromosome ratio. Example

[0208] The invention will now be further illustrated by the following non-limiting examples. Unless otherwise stated, standard techniques from molecular biology, cell culture, biochemistry, and nucleic acid chemistry are used. Materials and Methods

[0209] The following describes examples of the procedures used in accordance with this disclosure:

[0210] Apoptosis induction and cfDNA fragment release in adherent target cell lines

[0211] The target adherent cell lines were placed in 3 to 4 150 cm² cells. 2Culture the cells in appropriate serum-containing cell culture medium in the flasks to 90% to 100% confluence. Before DMSO treatment, aspirate the culture medium from each flask and replace it with 16 mL of 5% FBS medium containing DMSO at a DMSO to medium ratio of 1:5. Under standard conditions (37°C, 5% CO2, and 18.6% to 20.9% O2), incubate the target adherent cell lines with the DMSO-containing medium in a standard cell culture incubator for 4 hours. After incubation, collect the DMSO-containing supernatant / medium in 50 mL tubes and store at -70°C to -80°C, or process for DNA extraction.

[0212] Induction of apoptosis and release of cfDNA fragments in suspension target cell lines

[0213] The target suspension cell lines were placed in 2 to 3 150 cm⁻¹ spaces. 2 Incubate the cells in appropriate serum-containing cell culture medium in the flasks to 90-100% confluence. Prior to DMSO treatment, transfer each cell culture to a separate 50 mL conical tube and centrifuge at 500 RCF (xg) for 5 minutes to obtain a cell pellet. Aspirate the culture medium from each conical tube and replace it with 16 mL of 5% FBS medium containing DMSO at a DMSO to medium ratio of 1:5 per conical tube. Resuspend the cell pellet in DMSO-containing medium. Incubate the target suspension cell line with DMSO-containing medium in a standard cell culture incubator for 4 hours under standard conditions (37°C, 5% CO2, and 18.6% to 20.9% O2). After incubation, centrifuge each conical flask containing the target suspension cell line at 500 RCF (xg) for 5 minutes to obtain a cell pellet. Collect the DMSO-containing supernatant / medium in 50 mL tubes and store at -70°C to -80°C, or process by cfDNA extraction.

[0214] cfDNA extraction

[0215] cfDNA extraction was performed using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Cat # 55114) following the manufacturer's instructions.

[0216] cfDNA collection based on the US11753682B2 method

[0217] In short, treat selected cell lines with the ionotropic carrier monensin at a concentration of 5 to 15 µM for at least 1 day (24 hours) in a humid atmosphere of 37°C and 5% CO2. Then centrifuge the culture medium at 200 g for 30 minutes at room temperature. Remove the supernatant and mix with an equal volume of total exosome separation reagent. Incubate the cell culture medium overnight at 4°C. After incubation, centrifuge the culture medium at 10,000 g for 1 hour at 4°C. Discard the supernatant, resuspend the precipitate in PBS, and store at -80°C. cfDNA can be isolated from the extracellular vesicle precipitate using a silica membrane (e.g., the QIAGEN Cyclic Nucleic Acid Separation Kit).

[0218] Size selection

[0219] 0.8x + 1.5x size selection (for obtaining short clips and clips of the desired length)

[0220] Before beginning cfDNA size selection, equilibrate the AMPure XP beads at room temperature for 15 minutes, then vortex for a few seconds. Add 0.8 times the volume of the original extracted cfDNA sample to the extracted cfDNA and mix by briefly vortexing the cfDNA bead mixture. Incubate the cfDNA bead mixture at room temperature for 15 minutes, then transfer it to a magnetic rack and incubate for another 5 to 10 minutes until the mixture becomes clear and the beads aggregate to one side / corner of the container containing the mixture. Transfer the clear supernatant to a new container, reserving the original container containing the beads for fragment binding quality control. Add 1.5 times the volume of the original extracted cfDNA sample to the clear supernatant in the new container and mix the cfDNA bead mixture by briefly vortexing or pipetting. Incubate the cfDNA bead mixture at room temperature for 5 minutes, then transfer it to a magnetic rack and incubate for another 5 to 10 minutes until the mixture becomes clear and the beads aggregate to one side / corner of the container containing the mixture. Remove the clear supernatant. While in contact with the magnet, add 2 volumes of 70% ethanol to all containers containing beads, including the original container containing the first beads, and incubate at room temperature for 30 seconds. Then, remove the 70% ethanol from the containers while in contact with the magnet. While in contact with the magnet, add another 2 volumes of 70% ethanol to all containers containing beads and incubate at room temperature for 30 seconds. Then, remove the 70% ethanol from the containers while in contact with the magnet and air dry the beads for 5 to 10 minutes. Then, remove the containers containing beads from the magnet and add 1.0 volume of elution buffer relative to the original extracted cfDNA sample volume to all containers containing beads, and mix by pipetting. Then, incubate the containers containing the elution buffer bead mixture at room temperature for 5 minutes, then transfer them to the magnet and incubate for another 5 to 10 minutes, until the elution buffer microsphere mixture becomes clear and the beads aggregate on one side / corner of the container containing the elution buffer bead mixture. Then, transfer the clear supernatant to a new container.

[0221] Fragment size analysis

[0222] The fragment length distribution of cfDNA-like DNA fragments obtained by this method was analyzed using the High Sensitivity DNA Bioanalyzer Kit on the Agilent Bioanalyzer platform.

[0223] ddPCR

[0224] Chromosomal ratios of cfDNA-like DNA fragments were analyzed using dPCR aneuploidy assays with concentrations ranging from 1.5 to 10 ng. The homogeneity of target representation was assessed by calculating the ratios between target concentrations. Ratios of cfDNA-like DNA fragments within approximately ±30%, ±25%, ±20%, ±15%, or ±10% of the expected ratio, for example, within approximately ±20%, are considered to accurately reflect the distribution of native cfDNA and are used for further quality control. Those skilled in the art can use any method available for dPCR aneuploidy assays to evaluate cfDNA-like DNA fragments.

[0225] The following examples further illustrate various aspects and implementation schemes of the methods disclosed herein.

[0226] Example 1

[0227] like Figure 1 As shown in Figure A, 4 hours after treatment with the fibroblast 397 cell line, cisplatin treatment produced the lowest percentage of viable cells, approximately 28%, followed by monensin at approximately 41%, and DMSO at approximately 52%. At 12 hours post-treatment, the percentages of viable cells under DMSO and monensin conditions were approximately 23% and 31%, respectively, significantly lower than at 4 hours post-treatment. In contrast, the percentage of viable cells under cisplatin conditions was approximately 26%, generally comparable to the viability of cells treated with cisplatin at 4 hours. At 24 hours post-treatment, cell viability in each condition was generally comparable to that at 12 hours post-treatment.

[0228] For the lymphocyte 695 cell line, such as Figure 1 As shown in Figure B, at 4 hours post-treatment, cisplatin treatment produced the lowest percentage of viable cells, approximately 3%, followed by DMSO at approximately 24%, and monensin at approximately 39%. At 12 hours post-treatment, the percentages of viable cells under DMSO and monensin conditions were approximately 17% and 30%, respectively, significantly lower than at 4 hours post-treatment. In contrast, the percentage of viable cells under cisplatin conditions was approximately 9%. At 24 hours post-treatment, DMSO treatment produced the lowest percentage of viable cells, approximately 7%, followed by cisplatin at approximately 12%, and monensin at approximately 38%.

[0229] Example 2

[0230] Figure 2 The results showed that DMSO treatment of fibroblast 397 cells relatively increased the total DNA yield compared with treatment with monensin, cisplatin, or control media. Notably, the total DNA obtained from fibroblast 397 cells after 4 hours of DMSO treatment was relatively less than that after 12 and 24 hours of DMSO treatment.

[0231] In comparison, Figure 2This indicates that, at all post-treatment time points tested on the Lymphocyte 695 cell line, the culture medium treatment conditions produced relatively (substantially) higher DNA yields compared to all other conditions. Furthermore, at all post-treatment time points tested, the monensin treatment conditions produced relatively (substantially) higher DNA yields compared to the DMSO treatment conditions for the Lymphocyte 695 cell line.

[0232] Example 3

[0233] Figure 3 A and 3B demonstrate that the cfDNA-like DNA fragments generated by the method according to the present invention (apoptosis method) accurately capture the size distribution of the patient's cfDNA, with an expected average length of 170 bp and a similar distribution of approximately 170 bp.

[0234] In addition, such as Figure 3 As shown in B, the sample / DNA fragment ratio containing cfDNA-like DNA fragments is similar to the size distribution of the patient's cfDNA, or comparable to the cfDNA-like material generation method using monensin described in US11753682B2. Figure 3 In B, it is referred to as "BT," which is either superior to comparable methods for generating cfDNA-like materials.

[0235] In addition, such as Figure 3 As shown in B, compared with the method using monensin described in US11753682B2, the method according to the present invention yields a narrower distribution of cfDNA-like DNA fragments 4 hours after DMSO treatment, which is more similar to patient samples than the method in US117536822B2, which shows a wider fragment distribution than both patient samples and the DMSO apoptosis method.

[0236] Example 4

[0237] Figure 4 This indicates that a relatively high total DNA amount does not equate to a high amount of cfDNA-like DNA fragments with an ideal size distribution similar to that of the patient's cfDNA.

[0238] For example, such as Figure 2 As shown in B, although the total DNA yield produced by lymphocyte 695 cells 4 hours after culture medium treatment was higher than that of DMSO, it was still comparable to... Figure 4 Compared to the ideal cfDNA-like DNA fragment shown in C, the total DNA contains a greater amount of HMW DNA. Furthermore, as... Figure 2 As shown in B, although lymphocyte 695 cells produced a higher total DNA yield 4 hours after monensin treatment, such as Figure 4 As shown in C, this DNA is almost entirely HMW DNA, which is not ideal.

[0239] like Figure 4 As shown in Figure B, the size distribution of cfDNA produced 4 hours after DMSO treatment was comparable to that obtained from the patient, and almost no HMW fragments were produced in the cell types tested. Notably, the conditions 4 hours after DMSO treatment produced a distribution of cfDNA-like DNA fragments most similar to those in the patient samples.

[0240] It is worth noting that, Figure 4 The cfDNA-like DNA fragments involved in the data have not been size-selected, and therefore represent cfDNA-like DNA fragments obtained only from the post-processing of the culture medium.

[0241] Example 5

[0242] Figure 5 The results showed that the condition exhibiting chromosome ratios closest to all expected chromosome ratios in both euploid and aneuploid samples was the condition 4 hours after DMSO treatment, which showed an aneuploid Chr21 ratio of approximately 1.5 to all euploid chromosomes (see lane 2 – aneuploid fibroblast 397 cell line) and an euploid chromosome ratio of 1.0 to the euploid genome (see lane 2 – aneuploid fibroblast 997 cell line and lane 8 – euploid lymphocyte 695 cell line). Furthermore, compared to all other conditions, the variance of chromosome ratios in aneuploid cells treated with DMSO for 4 hours was reduced at 4 hours.

[0243] In contrast, aneuploid fibroblasts 397 (trisomy 21) did not show the expected chromosome 21 / 22q ratio of 1.5 under monensin treatment at all time points, producing inaccurate / misleading chromosome ratios.

[0244] It is worth noting that the 4-hour and 12-hour culture conditions do not produce ideal aneuploid ratios, especially for Chr21 / Chro22q and Chr21 / Chr13, where the ratio of approximately 1.75 is too high and therefore unfavorable. In this regard, using control samples with a chromosome ratio greater than 1.5 to create artificial samples mimicking a 4% fetal fraction sample is misleading, as it will produce a higher chromosome ratio than a real sample. For example, if used to test analytical capabilities, it will produce unrealistically good results and overestimate the intended detection capacity. Furthermore, Figure 5 This indicates that although the DNA fragments generated under these conditions have an ideal size distribution, they do not resemble the patient samples in aneuploidy detection and do not produce the expected chromosome ratios. This suggests that size distribution alone is insufficient to reproduce the characteristics of the patient cfDNA sample or produce the expected genome ratios.

[0245] In contrast, the conditions after 4 hours of DMSO treatment produced the expected chromosome ratios for both aneuploid and euploid cell lines. For example, for the 395 cell line (Chr21 aneuploidy), the chromosome ratios for Chr21 / 22q, Chr21 / 13, and Chr21 / 18 were approximately 1.5, and the chromosome ratio for Chr18 / 13 was approximately 1. For the 696 cell line (euploidy), all chromosome ratios were around 1.0, which was in line with expectations.

[0246] Terms and Conditions

[0247] The inventive concept is set forth in the following clauses:

[0248] Clause Group M (Method Clause):

[0249] M1. A method for generating multiple cfDNA-like DNA fragments, the method comprising:

[0250] i. Provide target cell lines;

[0251] ii. Contact the target cell line with a culture medium containing DMSO; and

[0252] iii. Incubate the target cell line with a medium containing DMSO to induce apoptosis in the target cell line and generate cfDNA-like DNA fragments.

[0253] M2. The method according to clause M1, wherein the target cell line is an adherent cell line.

[0254] M3. The method according to clause M1, wherein the target cell line is a suspension cell line.

[0255] M4. A method for generating multiple cfDNA-like DNA fragments from a target adherent cell line, the method comprising:

[0256] i. Provide targeted adherent cell lines;

[0257] ii. Remove the culture medium from the target adherent cell line;

[0258] iii. Contact the target adherent cell line with a culture medium containing DMSO;

[0259] iv. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0260] M5. A method for generating multiple cfDNA-like DNA fragments from a target adherent cell line, the method comprising:

[0261] i. Provide target adherent cell lines in the culture medium;

[0262] ii. Add DMSO to the culture medium in contact with the target adherent cell line;

[0263] iii. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0264] M6. A method for generating multiple cfDNA-like DNA fragments from a target suspension cell line, the method comprising:

[0265] i. Provide target suspension cell lines;

[0266] ii. Isolate cells from the target suspension cell line from the culture medium;

[0267] iii. Contact the isolated cells of the target suspension cell line with a culture medium containing DMSO;

[0268] iv. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0269] M6a. A method for generating multiple cfDNA-like DNA fragments from a target suspension cell line, the method comprising:

[0270] i. Provide the target suspension cell line in the culture medium;

[0271] ii. Add DMSO to the culture medium in contact with the target suspension cell line;

[0272] iii. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0273] M7. The method according to clause M6, wherein isolating cells of the target suspension cell line from the culture medium comprises:

[0274] The culture medium containing the target suspended cell line is separated by precipitation to obtain a culture supernatant and a precipitated target suspended cell line; and

[0275] Remove the culture medium supernatant from the precipitated target suspension cell line.

[0276] M8. The method according to clause M7, wherein the precipitated target suspension cell line is produced by centrifugation or gravity separation.

[0277] M9. The method according to any one of clauses M6, M7 or M8, wherein separating cells from the target suspension cell line from the culture medium comprises removing the culture medium supernatant from the precipitated target suspension cell line by aspiration.

[0278] M10. The method according to any one of clauses M6, M7, M8 or M9, wherein contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO comprises resuspending the precipitated cells of the target suspension cell line in the culture medium containing DMSO.

[0279] M11. The method according to any of the preceding clauses, wherein the target cell line is obtained by a cell sorting method, and the method includes contacting the sorted cells with a culture medium containing DMSO.

[0280] M11a. The method according to any one of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments, and the cfDNA-like nucleic acid fragments are released into a medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0281] M12. The method according to any of the preceding clauses further includes extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

[0282] M13. The method according to clause M12, wherein extracting the cfDNA-like DNA fragment from a culture medium containing DMSO and the cfDNA-like DNA fragment comprises:

[0283] Collect culture media containing DMSO and cfDNA-like DNA fragments; and

[0284] DNA extraction was performed on a culture medium containing DMSO and cfDNA-like DNA fragments to obtain a sample containing multiple cfDNA-like DNA fragments.

[0285] M14. The method of any of the preceding clauses includes obtaining a cfDNA-like DNA fragment of the desired length.

[0286] M14a. The method of any of the preceding clauses, wherein the cfDNA-like DNA fragment has the desired length.

[0287] M15. The method according to clause M14, wherein obtaining a cfDNA-like DNA fragment of the desired length includes size selection of the cfDNA-like DNA fragments in a sample containing multiple cfDNA-like DNA fragments.

[0288] M16. The method described in accordance with clause M15, wherein size selection includes single-core body size selection.

[0289] M17. The method according to any one of clauses M14 to M16, wherein the ideal length of the cfDNA-like DNA fragment is between about 50 bp and 300 bp, between about 100 bp and 250 bp, or between about 150 bp and 200 bp.

[0290] M18. The method according to any one of clauses M14 to M17, wherein the ideal length of the cfDNA-like DNA fragment is between about 150 bp and 170 bp; optionally about 160 bp.

[0291] M19. The method according to any of the preceding clauses, wherein the DMSO-containing culture medium contains about 40% to 1%, about 35% to 5%, about 30% to 10%, or about 25% to 15% DMSO.

[0292] M20. The method according to any of the preceding clauses, wherein the culture medium containing DMSO contains about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO.

[0293] M21. The method according to any of the preceding clauses, wherein the culture medium containing DMSO contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO.

[0294] M22. The method according to any of the preceding clauses, wherein the culture medium containing DMSO contains up to about 20% fetal bovine serum (FBS), up to about 10% FBS, up to about 9% FBS, up to about 8% FBS, up to about 7% FBS, up to about 6% FBS, up to about 5% FBS, up to about 4% FBS, up to about 3% FBS, up to about 2% FBS, up to about 1% FBS, or up to about 0.5% FBS.

[0295] M23. The method according to any of the preceding clauses, wherein the culture medium containing DMSO contains about 0.5% to 20% FBS, about 1% to 10% FBS, about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS.

[0296] M24. The method according to any of the preceding clauses, wherein the DMSO-containing culture medium comprises about 10% FBS, about 9% FBS, about 8% FBS, about 7% FBS, about 6% FBS, about 5% FBS, about 4% FBS, about 3% FBS, about 2% FBS, about 1% FBS, or about 0.5% FBS.

[0297] M25. The method according to any of the preceding clauses, wherein the DMSO-containing medium comprises about 40% DMSO and about 60% DMSO-containing medium containing 5% FBS, about 35% DMSO and about 65% DMSO-containing medium containing 5% FBS, about 30% DMSO and about 70% DMSO-containing medium containing 5% FBS, about 25% DMSO and about 75% DMSO-containing medium containing 5% FBS, about 20% DMSO and about 80% DMSO-containing medium containing 5% FBS, about 15% DMSO and about 85% DMSO-containing medium containing 5% FBS, about 10% DMSO and about 90% DMSO-containing medium containing 5% FBS, about 5% DMSO and about 95% DMSO-containing medium containing 5% FBS, or about 1% DMSO and about 99% DMSO-containing medium containing 5% FBS.

[0298] M26. The method according to any of the preceding clauses, wherein the DMSO-containing medium comprises about 25% DMSO and about 75% DMSO containing 5% FBS, about 24% DMSO and about 76% DMSO containing 5% FBS, about 23% DMSO and about 77% DMSO containing 5% FBS, about 22% DMSO and about 78% DMSO containing 5% FBS, about 21% DMSO and about 79% DMSO containing 5% FBS, about 20% DMSO and about 80% DMSO containing 5% FBS, about 19% DMSO and about 81% DMSO containing 5% FBS, about 18% DMSO and about 82% DMSO containing 5% FBS, about 17% DMSO and about 83% DMSO containing 5% FBS, about 16% DMSO and about 84% DMSO containing 5% FBS, or about 15% DMSO and about 85% DMSO containing 5% FBS.

[0299] M27. The method according to any of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours, or up to about 4 hours.

[0300] M28. The method according to any of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

[0301] M29. The method according to any of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO for about 0.5, about 1, about 2, about 3, or about 4 hours.

[0302] M29a. The method according to any of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO for about 0.5 to 8 hours, about 0.5 to 7 hours, about 1 to 7 hours, about 1 to 6 hours, about 2 to 6 hours, about 2 to 5 hours, about 2.5 to 5 hours, about 2.5 to 4.5 hours, about 3 to 4.5 hours, or about 3 to 4 hours.

[0303] M30. The method according to any of the preceding clauses, wherein the target cell line is incubated with a medium containing DMSO at a temperature of about 37°C.

[0304] M31. The method described under any of the preceding clauses includes determining copy number variability of DNA regions in a target cell line based on the presence of cfDNA-like DNA fragments.

[0305] M32. The method described in any of the preceding clauses includes performing a dPCR reaction or a qPCR reaction.

[0306] M33. The method according to clause M32, wherein dPCR or qPCR is performed on cfDNA-like DNA fragments to determine the fetal fraction of the biological sample.

[0307] M34. The method according to any of the preceding clauses, wherein dPCR or qPCR is performed on cfDNA-like DNA fragments to obtain the relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region.

[0308] M35. The method according to clause M34, wherein the relative abundance of the first chromosome relative to the second chromosome, or the relative abundance of the first genomic region relative to the second genomic region, comprises a first ratio between the first chromosome and the second chromosome or between the first genomic region and the second genomic region, wherein suitably, the relative abundance of the first chromosome relative to the second chromosome comprises a first chromosome ratio.

[0309] M36. The method according to clause M34 or M35, wherein the first chromosome or the first genomic region is known or expected to be aneuploid in the target cell line, and the second chromosome or the second genomic region is known or expected to be euploid in the target cell line.

[0310] M37. The method according to any one of clauses M34 to M36, wherein the first chromosome or the first genomic region is known or expected to be triploid in the target cell line, and the second chromosome or the second genomic region is known or expected to be diploid in the target cell line, suitably wherein the target cell line is a target adherent cell line or a target suspension cell line.

[0311] M38. The method described in accordance with clause M35, or M36 or M37 when attached to clause M35, wherein the first chromosome ratio is in the range of about 1.40 to 1.60, about 1.42 to 1.58, about 1.44 to 1.56, about 1.46 to 1.54 or about 1.48 to 1.52, for example about 1.5.

[0312] M39. The method according to clause M34 or M35, wherein the first chromosome is known or expected to be euploid in the target cell line, the second chromosome is known or expected to be euploid in the target cell line, and suitably wherein the target cell line is a target adherent cell line or a target suspension cell line.

[0313] M40. The method according to any one of clauses M34, M35 or M39, wherein the first chromosome is known or expected to be diploid in the target cell line and the second chromosome is known or expected to be diploid in the target cell line.

[0314] M41. The method described in accordance with clause M35, or M39 or M40 when attached to clause M35, wherein the first chromosome ratio is about 0.9-1.1, about 0.92-1.08, about 0.94-1.06, about 0.96-1.04, about 0.98-1.02, for example about 1.0.

[0315] M42. The method described in accordance with Clause M35, or M36 to M41, when dependent on Clause M35, wherein the first ratio of cfDNA-like DNA fragments is the same, substantially the same, or similar to the ratio obtained from a patient's cfDNA sample, and suitably, wherein the first chromosome ratio of cfDNA-like DNA fragments is the same, substantially the same, or similar to the chromosome ratio obtained from a patient's cfDNA sample.

[0316] M43. The method described under clause M35, or M36 to M41, when dependent on clause M35, wherein the first ratio of cfDNA-like DNA fragments differs from the ratio obtained from the patient's cfDNA sample by up to about 15%, for example, by up to about 12.5%, up to about 10%, up to about 7.5%, up to 5%, up to 2.5%, or up to about 1% compared to the chromosome ratio from the patient's cfDNA sample.

[0317] M43a. The method described in accordance with Clause M35, or M36 to M41, when attached to Clause M35, wherein the first ratio of the cfDNA-like DNA fragment differs from the ratio obtained from the patient's cfDNA sample by about -10% to +10%, for example, about -8% to +8%, about -6% to +6%, about -4% to +4%, or about -2% to +2%.

[0318] M43b. The method according to clause M43 or M43a, wherein the first ratio is a first chromosome ratio.

[0319] M44. A method for preparing multiple cfDNA-like DNA fragments, the method comprising:

[0320] i. Provide target cell lines

[0321] ii. Lyse the target cell line cells with a buffer containing ATAC-RSB to obtain target cell line DNA; and

[0322] iii. Digest the DNA of the target cell line with micrococcal nuclease to produce cfDNA-like DNA fragments.

[0323] Terms and Conditions P (Product Terms and Conditions):

[0324] P1. Multiple cfDNA-like DNA fragments generated by the following methods:

[0325] i. Provide target cell lines;

[0326] ii. Contact the target cell line with a culture medium containing DMSO; and

[0327] iii. Incubate the target cell line with a medium containing DMSO to induce apoptosis in the target cell line and generate multiple cfDNA-like DNA fragments.

[0328] P2. The plurality of cfDNA-like DNA fragments as described in Clause P1, wherein the target cell line is an adherent cell line.

[0329] P3. The plurality of cfDNA-like DNA fragments as described in Clause P1, wherein the target cell line is a suspension cell line.

[0330] P4. Multiple cfDNA-like DNA fragments generated by the following methods:

[0331] i. Provide targeted adherent cell lines;

[0332] ii. Remove the culture medium from the target adherent cell line;

[0333] iii. Contact the target adherent cell line with a culture medium containing DMSO;

[0334] iv. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0335] P5. Multiple cfDNA-like DNA fragments were generated using the following methods:

[0336] iv. Provide target adherent cell lines in the culture medium;

[0337] v. Add DMSO to the culture medium in contact with the target adherent cell line;

[0338] vi. Incubate target adherent cell lines with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0339] P6. Multiple cfDNA-like DNA fragments were generated using the following methods:

[0340] i. Provide target suspension cell lines;

[0341] ii. Isolate cells from the target suspension cell line from the culture medium;

[0342] iii. Contact the isolated cells of the target suspension cell line with a culture medium containing DMSO;

[0343] iv. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0344] P6a. Multiple cfDNA-like DNA fragments prepared by the following method:

[0345] i. Provide the target suspension cell line in the culture medium;

[0346] ii. Add DMSO to the culture medium in contact with the target suspension cell line;

[0347] iii. Incubate isolated cells of the target suspension cell line with a medium containing DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

[0348] P7. The plurality of cfDNA-like DNA fragments described in clause P6, wherein the cells from which the target suspension cell line was isolated from the culture medium include:

[0349] The culture medium containing the target suspended cell line is separated by precipitation to obtain a culture supernatant and a precipitated target suspended cell line; and

[0350] Remove the culture medium supernatant from the precipitated target suspension cell line.

[0351] P8. Multiple cfDNA-like DNA fragments as described in Clause P7, wherein the target suspension cell line of the precipitate is produced by centrifugation or gravity separation.

[0352] P9. A plurality of cfDNA-like DNA fragments according to any one of clauses P6, P7 or P8, wherein the isolation of cells from the target suspension cell line from the culture medium includes removing the culture medium supernatant from the precipitated target suspension cell line by aspiration.

[0353] P10. A plurality of cfDNA-like DNA fragments according to any one of clauses P6, P7, P8 or P9, wherein contacting the isolated cells of the target suspension cell line with a culture medium containing DMSO comprises resuspending the precipitated cells of the target suspension cell line in a cell culture medium containing DMSO.

[0354] P11. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P10, wherein cells of a target cell line are incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments, and the cfDNA-like DNA fragments are released into a medium containing DMSO, thereby generating a medium containing DMSO and cfDNA-like DNA fragments.

[0355] P12. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P11, the method further comprising extracting the cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like nucleic acid fragments.

[0356] P13. A plurality of cfDNA-like DNA fragments as described in clause P12, wherein extraction of the cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like nucleic acid fragments includes:

[0357] Collect culture media containing DMSO and cfDNA-like DNA fragments; and

[0358] DNA extraction was performed on a culture medium containing DMSO and cfDNA-like DNA fragments to obtain a sample containing multiple cfDNA-like DNA fragments.

[0359] P14. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P13, wherein the cfDNA-like DNA fragments have the desired length.

[0360] P15. The method further includes, according to Clause P14, selecting the size of the cfDNA-like DNA fragments in a sample containing the plurality of cfDNA-like DNA fragments to obtain cfDNA-like DNA fragments of desired length.

[0361] P16. Multiple cfDNA-like DNA fragments as described in clause P15, wherein size selection includes mononuclear body size selection.

[0362] P17. A plurality of cfDNA-like DNA fragments according to any one of clauses P14 to P16, wherein the ideal length of said cfDNA-like DNA fragments is between about 50 bp and 300 bp, between about 100 bp and 250 bp, or between about 150 bp and 200 bp.

[0363] P18. A plurality of cfDNA-like DNA fragments according to any one of clauses P14 to P17, wherein the ideal length of said cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally about 160 bp.

[0364] P19. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P18, wherein the DMSO-containing culture medium contains about 40% to 1%, about 35% to 5%, about 30% to 10%, or about 25% to 15% DMSO.

[0365] P20. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P19, wherein the DMSO-containing culture medium contains about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO.

[0366] P21. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P20, wherein the DMSO-containing culture medium contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO.

[0367] P22. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P21, wherein the DMSO-containing culture medium contains up to about 20% fetal bovine serum (FBS), up to about 10% FBS, up to about 9% FBS, up to about 8% FBS, up to about 7% FBS, up to about 6% FBS, up to about 5% FBS, up to about 4% FBS, up to about 3% FBS, up to about 2% FBS, up to about 1% FBS, or up to about 0.5% FBS.

[0368] P23. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P22, wherein the DMSO-containing culture medium contains about 0.5% to 20% FBS, about 1% to 10% FBS, about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS.

[0369] P24. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P23, wherein the DMSO-containing culture medium comprises about 10% FBS, about 9% FBS, about 8% FBS, about 7% FBS, about 6% FBS, about 5% FBS, about 4% FBS, about 3% FBS, about 2% FBS, about 1% FBS, or about 0.5% FBS.

[0370] P25. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P24, wherein the DMSO-containing medium comprises about 40% DMSO and about 60% containing 5% FBS, about 35% DMSO and about 65% containing 5% FBS, about 30% DMSO and about 70% containing 5% FBS, about 25% DMSO and about 75% containing 5% FBS, about 20% DMSO and about 80% containing 5% FBS, about 15% DMSO and about 85% containing 5% FBS, about 10% DMSO and about 90% containing 5% FBS, about 5% DMSO and about 95% containing 5% FBS, or about 1% DMSO and about 99% containing 5% FBS.

[0371] P26. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P25, wherein the DMSO-containing medium comprises about 25% DMSO and about 75% containing 5% FBS, about 24% DMSO and about 76% containing 5% FBS, about 23% DMSO and about 77% containing 5% FBS, about 22% DMSO and about 78% containing 5% FBS, about 21% DMSO and about 79% containing 5% FBS, about 20% DMSO and about 80% containing 5% FBS, about 19% DMSO and about 81% containing 5% FBS, about 18% DMSO and about 82% containing 5% FBS, about 17% DMSO and about 83% containing 5% FBS, about 16% DMSO and about 84% containing 5% FBS, or about 15% DMSO and about 85% containing 5% FBS.

[0372] P27. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P26, wherein the cells of the target cell line are incubated with a medium containing DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours, or up to about 4 hours.

[0373] P28. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P27, wherein the cells of the target cell line are incubated with a medium containing DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

[0374] P29. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P28, wherein the cells of the target cell line are incubated with a medium containing DMSO for about 0.5, about 1, about 2, about 3, or about 4 hours.

[0375] P30. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P29, wherein the cells of the target cell line are incubated with a medium containing DMSO for about 0.5 to 8 hours, about 0.5 to 7 hours, about 1 to 7 hours, about 1 to 6 hours, about 2 to 6 hours, about 2 to 5 hours, about 2.5 to 5 hours, about 2.5 to 4.5 hours, about 3 to 4.5 hours, or about 3 to 4 hours.

[0376] P31. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P30, wherein the target cell line cells are incubated with a medium containing DMSO at a temperature of about 37°C.

[0377] P32. A plurality of cfDNA-like DNA fragments according to any one of clauses P1 to P31, including determining copy number variability of DNA regions in a target cell line based on the presence of cfDNA-like DNA fragments.

[0378] P33. Multiple cfDNA-like DNA fragments are generated through the following process:

[0379] i. Provide target cell lines

[0380] ii. Lyse the target cell line cells with a buffer containing ATAC-RSB to obtain target cell line DNA; and

[0381] iii. Use micrococcal nuclease to break down the DNA of the target cell line to produce cfDNA-like DNA fragments.

Claims

1. A method for preparing multiple cfDNA-like DNA fragments, the method comprising: i. Provide target cell lines; ii. Contact the target cell line with a culture medium containing DMSO; and iii. Incubate the target cell line with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments.

2. The method according to claim 1, wherein the target cell line is incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments, and the cfDNA-like DNA fragments are released into a medium containing DMSO to generate a medium containing DMSO and cfDNA-like DNA fragments.

3. The method of claim 2 further comprises extracting cfDNA-like DNA fragments from a culture medium containing DMSO and cfDNA-like DNA fragments.

4. The method according to any one of the preceding claims, wherein the cfDNA-like DNA fragment has the desired length, or wherein the method further comprises obtaining a cfDNA-like DNA fragment having the desired length.

5. The method of claim 4, wherein the desired length of the cfDNA-like DNA fragment is about 150 bp to 170 bp; optionally about 160 bp.

6. The method according to any one of claims 1, 2 or 3, wherein the DMSO-containing culture medium comprises about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16% or about 15% DMSO.

7. The method according to any one of claims 1, 2 or 3, wherein the target cell line is incubated with a medium containing DMSO for about 0.5 to 8 hours, about 0.5 to 7 hours, about 1 to 7 hours, about 1 to 6 hours, about 2 to 6 hours, about 2 to 5 hours, about 2.5 to 5 hours, about 2.5 to 4.5 hours, about 3 to 4.5 hours or about 3 to 4 hours.

8. The method according to any one of claims 1, 2 or 3, wherein the DMSO-containing culture medium comprises about 0.5% to 20% FBS, about 1% to 10% FBS, or about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS or about 5% to 6% FBS.

9. The method of claim 1, wherein the cfDNA-like DNA fragment is subjected to dPCR or qPCR to obtain the relative abundance of a first chromosome relative to a second chromosome, or the relative abundance of a first genomic region relative to a second genomic region; optionally, wherein the relative abundance of a first chromosome relative to a second chromosome, or the relative abundance of a first genomic region relative to a second genomic region, includes a first chromosome ratio of the first chromosome to the second chromosome; optionally, wherein the first chromosome ratio of the cfDNA-like DNA fragment is the same, substantially the same, or similar to the chromosome ratio obtained from a patient cfDNA sample.

10. The method of claim 9, wherein the first chromosome or the first genomic region is known or expected to be aneuploid in the target cell line, and the second chromosome or the second genomic region is known or expected to be euploid in the target cell line.

11. The method of claim 9 or 10, wherein the first chromosome ratio of the cfDNA-like DNA fragment differs from the chromosome ratio obtained from the patient's cfDNA sample by up to about 15%, for example, by up to about 12.5%, up to about 10%, up to about 7.5%, up to about 5%, up to about 2.5%, or up to about 1%, for example, up to about 10%.

12. The method of claim 9 or 10, wherein the first chromosome ratio is between about 1.40 and 1.60, between about 1.42 and 1.58, between about 1.44 and 1.56, between about 1.43 and 1.54, or between about 1.48 and 1.52, for example, about 1.

5.

13. Multiple cfDNA-like DNA fragments were generated using the following methods: i. Provide target cell lines; Ii. Contact the target cell line with a culture medium containing DMSO; and iii. Incubate the target cell line with a medium containing DMSO to induce apoptosis in the target cell line and generate multiple cfDNA-like DNA fragments.

14. The plurality of cfDNA-like DNA fragments according to claim 13, wherein the target cell line is incubated with a medium containing DMSO to induce apoptosis of the target cell line and generate cfDNA-like DNA fragments, and the cfDNA-like DNA fragments are released into the medium containing DMSO to generate a medium containing DMSO and cfDNA-like DNA fragments.

15. The method of claim 13 or 14, comprising extracting the cfDNA-like DNA fragments from a culture medium containing DMSO and the cfDNA-like DNA fragments.

16. The plurality of cfDNA-like DNA fragments according to claim 13 or 14, wherein the cfDNA-like DNA fragments have the desired length.

17. The plurality of cfDNA-like DNA fragments according to claim 13 or 14, wherein the desired length of said cfDNA-like DNA fragment is about 150 bp to 170 bp; optionally about 160 bp.

18. The plurality of cfDNA-like DNA fragments according to claim 13 or 14, wherein the DMSO-containing culture medium contains about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, or about 15% DMSO.

19. The plurality of cfDNA-like DNA fragments according to claim 13 or 14, wherein the DMSO-containing culture medium comprises about 0.5% to 20% FBS, about 1% to 10% FBS, or about 2% to 9% FBS, about 3% to 8% FBS, about 4% to 7% FBS, or about 5% to 6% FBS.

20. The plurality of cfDNA-like DNA fragments according to claim 13 or 14, wherein the target cell line cells are incubated with a medium containing DMSO for about 0.5 to 8 hours, about 0.5 to 7 hours, about 1 to 7 hours, about 1 to 6 hours, about 2 to 6 hours, about 2 to 5 hours, about 2.5 to 5 hours, about 2.5 to 4.5 hours, about 3 to 4.5 hours, or about 3 to 4 hours.

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