WO2025151472A1

WO2025151472A1 - Methods for generating cfdna-like dna fragments

Info

Publication number: WO2025151472A1
Application number: PCT/US2025/010671
Authority: WO
Inventors: Maria F. GENCOGLU; Severine MARGERIDON
Original assignee: Bio Rad Laboratories Inc
Current assignee: Bio Rad Laboratories Inc
Priority date: 2024-01-08
Filing date: 2025-01-08
Publication date: 2025-07-17
Anticipated expiration: 2026-07-08

Abstract

Disclosed herein are cfDNA-like DNA fragments and broadly applicable methods to prepare cfDNA-like DNA fragments which produce a high yield and/or large quantities of cfDNA-like DNA fragments. The cfDNA-like DNA fragments produced according to the methods of the disclosure accurately capture the size distribution and diagnostic assay performance of natural cfDNA, and may therefore be used for assay methodological validation, internal assay quality control and external assay quality evaluation with high reproducibility and consistency.

Description

METHODS FOR GENERATING cfDNA-LIKE DNA FRAGMENTS

FIELD OF THE INVENTION

The present disclosure relates to cell-free DNA (cfDNA), and methods for the production of cfDNA; particularly for use in assay development, such as non-invasive prenatal testing (NIPT) assays.

BACKGROUND OF THE INVENTION

Many common human diseases are associated with mutations present in the human genome, of which a significant proportion are heritable or may occur during fertilisation or early embryonic development. Early detection of particular conditions or diseases in humans is desirable, such as foetal chromosomal abnormalities, enabling clinical/therapeutic interventions which may result in an improved prognosis for the patient.

Screening for disease-associated genetic perturbations is routine practice in healthcare, the most common example being non-invasive prenatal testing (NIPT), comprising prenatal screening for trisomy 21 , 18, and 13. However, currently available diagnostic assays for early detection of diseases that yield a diagnosis, and not merely a screening for a condition or disease, typically require an invasive procedure (e.g. amniocentesis or chorionic villus sampling), which have an increased risk of adverse reactions, such as infection, infection transmission, Rhesus sensitisation, premature labour, and miscarriage. Additionally, these invasive procedures are expensive and highly technical, and as such are not adapted for widespread use. Therefore, there is a need to develop improved NIPT diagnostic assays.

Current NIPT methods assay cell-free DNA (cfDNA), of which a small fraction in maternal blood is of foetal origin, to determine whether the foetal cfDNA component is indicative of one or more genetic abnormalities. cfDNA is typically comprised of unstable and short-lived small DNA molecules around 170 base pairs (bp) in length, which are present at low levels in maternal whole blood, e.g. 0-100 ng/mL. Furthermore, whilst the median foetal fraction of cfDNA is around 11% in the first trimester of pregnancy, it can be lower 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, particularly foetal cfDNA, clinical samples are highly valuable research materials, but are limited in their availability. Consequently, one of the major bottlenecks in NIPT diagnostic assay development is the availability of reference cfDNA samples, as the ideal reference cfDNA samples are derived from clinical samples. Thus, it is desirable to develop cfDNA-like reference samples to replace clinical samples with assay performance consistent with clinical samples.

Significant research efforts have been made to develop methods to produce cfDNA-like samples which perform similarly to a natural cfDNA sample. However, the development of cfDNA-like samples is complicated by the size distributions and fragmentation patterns present in natural cfDNA. Whilst the exact mechanisms of cfDNA formation have yet to be fully elucidated, the size distribution of cfDNA closely resembles that of DNA degraded during apoptosis, although cfDNA production may not necessarily be contingent on apoptosis. Many of these methods to obtain cfDNA-like fragments relate to obtaining short DNA fragments having a length similar to that of cfDNA, e.g. about 150 to 200 bp, from shearing longer genomic DNA by sonication or enzymatic digestion. However, cfDNA-like fragments that are derived from sonicated or enzymatic digested DNA often have significant limitations. It has been reported that the fragmentation characteristics of cell-free DNA from plasma can differ significantly from that of genomic DNA, yielding DNA fragments which differ in their sequence information and fragment length to natural cfDNA. cfDNA-like fragments generated by these methods typically perform poorly in NIPT assays, leading to erroneous conclusions, and limiting their use in assay development.

Having regard to the prior art, therefore, it would be desirable to have further methods for generating cfDNA (or cfDNA-like fragments) with properties similar to natural cfDNA which perform similarly to natural cfDNA in diagnostic assays. Such methods may be particularly beneficial for use as NIPT assay reference material, and in the development of NIPT assays, which may comprise dPCR or qPCR assays.

The present invention seeks to overcome one or more of the problems found in the prior art.

SUMMARY OF THE INVENTION

The present invention relates to one or more methods for the production of cfDNA; particularly for use in assay development, such as non-invasive prenatal testing (NIPT) assays. The methods of the invention are beneficial in producing a desirable balance between the quantity of cfDNA that can be obtained from a biological I cell sample; and the size distribution of the cfDNA fragments obtained. In particular, the cfDNA samples obtained by the methods of the invention provide improved performance in mimicking naturally derived cfDNA samples over alternative methods of obtaining cfDNA-containing material artificially. In one aspect, there is provided a method for generating a plurality of cfDNA-like DNA fragments, the method comprising: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments.

In another aspect, there is provided a method for generating a plurality of 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 medium comprising DMSO; iv. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

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

In another aspect, there is provided a method for generating a plurality of cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing a target suspension cell line; ii. separating cells of the target suspension cell line from a culturing medium; iii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iv. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In another aspect, there is provided a method for generating a plurality of cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target suspension cell line; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In embodiments of these aspects, incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments. In embodiments, the method may further comprise extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

Beneficially, according to embodiments, the cfDNA-like DNA fragments have a desired length. In embodiments, therefore, the method further comprises obtaining cfDNA-like DNA fragments having a desired length. Suitably, the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp, such as, about 160 bp.

In embodiments, the medium comprising DMSO 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.

According to embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for between about 0.5 and 8 hours, between about 0.5 and 7 hours, between about 1 and 7 hours, between about 1 and 6 hours, between about 2 and 6 hours, between about 2 and 5 hours, between about 2.5 and 5 hours, between about 2.5 and 4.5 hours, between about 3 and 4.5 hours, or between about 3 and 4 hours.

In embodiments, the medium comprising DMSO may further comprise between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS.

According to embodiments, dPCR or qPCR may be performed on the cfDNA-like DNA fragments to obtain a relative abundance of a first chromosome compared to a second chromosome, or a first genomic region compared to a second genomic region. The relative abundance of a first chromosome compared to a second chromosome, or the relative abundance of a first genomic region compared to a second genomic region, may be determined by a first chromosome ratio between the first chromosome and the second chromosome. The first chromosome ratio of the cfDNA-like DNA fragments is beneficially identical to, substantially the same as, or similar to a chromosome ratio obtained from a natural patient cfDNA sample.

In embodiments, 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.

In embodiments, the first chromosome ratio of the cfDNA-like DNA fragments may suitably be about 15% different to a chromosome ratio obtained from a patient cfDNA sample, such as about 12.5%, about 10%, about 7.5%, about 5%, about 2.5%, or about 1% different to a chromosome ratio from a patient cfDNA sample, e.g. about 10%.

In embodiments, the first chromosome ratio may be between about 1 .40 to 1 .60, between about 1.42 to 1.58, between about 1.44 to 1.56, between about 1.46 to 1.54, or between about 1.48 to 1.52, e.g. preferably about 1.5.

In another aspect, there is provided a method for generating a plurality of cfDNA-like DNA fragments, the method comprising: i. providing a target cell line ii. lysing cells of the target cell line with a buffer comprising ATAC-RSB to obtain DNA of the target cell line; and iii. digesting the DNA of the target cell line with Mnase to generate cfDNA-like DNA fragments.

In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of any aspect and/or embodiment of this disclosure. For example, produced by a method comprising: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate the plurality of cfDNA-like DNA fragments.

In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of: 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 medium comprising DMSO; iv. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target adherent cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target adherent cell line; iii. incubating the cells of the target adherent cell line with the culture medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target suspension cell line; ii. separating cells of the target suspension cell line from a culturing medium; iii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iv. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments. In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target suspension cell line; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In embodiments of these aspects, incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

In embodiments, the process for obtaining the plurality of cfDNA-like DNA fragments further comprises extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

According to embodiments, the cfDNA-like DNA fragments have a desired length. For example, the desirable length of the cfDNA-like DNA fragments may be between about 150 bp and 170 bp; particularly about 160 bp.

In embodiments, the plurality of cfDNA-like DNA fragments may be obtained by a method wherein the medium comprising DMSO 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. In embodiments, the medium comprising DMSO may further comprises between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS. In embodiments, to obtain the plurality of cfDNA-like DNA fragments, the cells of the target cell line may be incubated with the medium comprising DMSO for between about 0.5 and 8 hours, between about 0.5 and 7 hours, between about 1 and 7 hours, between about 1 and 6 hours, between about 2 and 6 hours, between about 2 and 5 hours, between about 2.5 and 5 hours, between about 2.5 and 4.5 hours, between about 3 and 4.5 hours, or between about 3 and 4 hours.

In another aspect, there is provided a plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target cell line ii. lysing cells of the target cell line with a buffer comprising ATAC-RSB to obtain DNA of the target cell line; and iii. digesting the DNA of the target cell line with Mnase to generate cfDNA-like DNA fragments. It will be appreciated that any features of any aspect or embodiment of the invention may be combined with any combination of features in any other aspect or embodiment of the invention, unless otherwise stated, and such combinations are specifically intended to fall within the scope of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the nature and advantages of embodiments of the present invention may be gained with reference to the following detailed description and the accompanying drawings. Those skilled in the art will appreciate that the invention described in the drawings is susceptible to variations and modifications other than those specifically described in the drawings. It is to be understood that the invention includes all such variations and modifications. The present invention is not to be limited in scope by the specific drawings described herein, which are intended for the purpose of exemplification only.

Figure 1 Different treatment conditions induce varying levels of apoptosis

Cell viability results obtained by treating cells with DMSO, monesin, cisplatin, or control Media. (A) Viability of cells from a Fibroblast cell line AG05397 (obtained from Coriell; ‘Fibroblast 397’), in response to treatment with DMSO, monesin, cisplatin, or control Media at 4 hours posttreatment, 12 hours post-treatment, and 24 hours post-treatment. The AG05397 cell line is from a male subject exhibiting Trisomy 21 condition. (B) Viability of cells from a B-lymphocyte cell line GM24695 (obtained from Coriell; ‘Lymphocyte 695’), in response to treatment with DMSO, monesin, cisplatin, or control Media at 4 hours post-treatment, 12 hours post-treatment, and 24 hours post-treatment. The GM24695 cell line is from a healthy, euploid female subject.

Figure 2 Different treatment conditions to generate cfDNA-like DNA fragments affect total DNA yield

Total DNA yield results obtained by treating cells with DMSO, monesin, cisplatin, or control Media. (A) Total DNA obtained from Fibroblast 397’s in response to treatment with DMSO, monesin, cisplatin, or control Media at 4 hours post-treatment, 12 hours post-treatment, and 24 hours post-treatment. (B) Total DNA obtained from Lymphocyte 695’s in response to treatment with DMSO, monesin, cisplatin, or control Media at 4 hours post-treatment, 12 hours posttreatment, and 24 hours post-treatment.

Figure 3 The size distribution of cfDNA from patients is more accurately captured by the cfDNA-like DNA fragments obtained according to the methods of the invention than comparative methods known in the art Comparison of size distribution of cfDNA fragments obtained from patient samples to cfDNA- like DNA fragments obtained according to the methods of the invention and comparative methods of the prior art. (A) Electrophoretogram of the size distribution of cfDNA samples obtained from patients by either plasmapheresis or streck tube and to two cfDNA-like DNA fragment samples obtained at 4 hours post DMSO treatment according to the methods of the invention. Approximately 58.2% of the DNA fragments obtained from the patient sample obtained by plasmapheresis exhibit a size distirubtion comensurate with the expected size distribution of cfDNA fragments. Approximately 72.2% of the DNA fragments obtained from the patient sample obtained by streck tube exhibit a size distirubtion commensurate with the expected size distribution of cfDNA fragments. ‘Sample 1 ’ - approximately 77.4% of the cfDNA- like DNA fragments obtained according to the methods of the invention exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments. ‘Sample 2’ - approximately 81.5% of the cfDNA-like DNA fragments obtained according to the methods of the invention exhibit a size distrbution comensurate with the expected size distribution of cfDNA fragments. (B) Electrophoretogram of the size distribution of cfDNA samples obtained from patient plasma bags by streck tube, or obtained at 4 hours post DMSO treatment according to the methods of the invention for the cell types 472M and Fibroblast 397 or methods disclosed in US11753682B2 (BT). Approximately 77.4% of the cfDNA-like DNA fragments obtained from 472M cells according to the methods of the invention exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments. Approximately 81 .5% of the cfDNA-like DNA fragments obtained from Fibroblast 397’s according to the methods of the invention exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments. Approximately 93.1% of the cfDNA-like DNA fragments obtained from a cfDNA sample (#t13) obtained from a patient sample according to the methods of US11753682B2 exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments. This sample exhibits the Trisomy 13 aneuploidy condition. The distribution of the cfDNA-like DNA fragments obtained by the method of US11753682B2 is comparatively wider compared to both the methods of the invention and the DNA fragments obtained from patient plasma bags by streck tube. Approximately 84.2% of the cfDNA-like DNA fragments obtained from a patient derived cfDNA sample (#xo) according to the methods of US11753682B2 exhibit a size distribution comensurate with the expected size distribution of cfDNA fragments. This sample exhibits the Monosomy X (Turner’s syndrome) aneuploidy condition. Again, the distribution of the cfDNA- like DNA fragments obtained by the method of US11753682B2 is comparatively wider compared to both the methods of the invention and the DNA fragments obtained from patient plasma bags by streck tube. Approximately 59.2% of the DNA fragments obtained by streck tube from a human plasma biological control sample from a female subject (#f7) exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments. Approximately 72.2% of the DNA fragments obtained by streck tube from a human plasma biological control sample from a male subject (#726M) exhibit a size distribution commensurate with the expected size distribution of cfDNA fragments.

Figure 4 Different treatment conditions to generate cfDNA-like DNA fragments affect the size distribution of cfDNA-like DNA fragments

Comparison of size distribution of cfDNA-like DNA fragments obtained in response to treatment with DMSO, monesin, or control media prior to any size-selection of the cfDNA-like DNA fragments. The peak of fragments obtained which are comensurate with the expected size distribution of cfDNA fragments are indicated by an arrow, around approximately 172 bp. (A) Electrophoretogram of the size distribution of cfDNA samples obtained from Fibroblast 397 cells and Lymphocyte 695 cells in response to treatment with monesin at 4 hours post-treatment, 12 hours post-treatment, and 24 hours post-treatment according to the methods of US11753682B2. At 4 hours post-treatment, the cfDNA-like DNA fragments obtained from Fibroblast 397 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. At 4 hours post-treatment, the cfDNA-like DNA fragments obtained from Lymphocyte 695 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. At 8 hours and 24 hours, DNA obtained from Lymphocytes 695 cells exhibits a significant peak having a size distribution commensurate with the expected size distribution of high molecular weight gDNA. (B) Electrophoretogram of the size distribution of cfDNA samples obtained from Fibroblast 397 cells and Lymphocyte 695 cells in response to treatment with DMSO at 4 hours post-treatment, 12 hours post-treatment, and 24 hours posttreatment according to the methods of the invention. At 4 hours post-treatment, the cfDNA-like DNA fragments obtained from Fibroblast 397 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. At 4 hours post-treatment, the cfDNA- like DNA fragments obtained from Lymphocyte 695 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. (C) Electrophoretogram of the size distribution of cfDNA samples obtained from Fibroblast 397 cells and Lymphocyte 695 cells in response to treatment with control media at 4 hours post-treatment, 12 hours post-treatment, and 24 hours post-treatment according to comparative methods to those of the invention. At 4 hours post-treatment, the cfDNA-like DNA fragments obtained from Fibroblast 397 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. At 4 hours post-treatment, the cfDNA-like DNA fragments obtained from Lymphocyte 695 cells exhibit a peak commensurate with the expected size distribution of cfDNA, e.g. around 172bp. At 4 hours, 8 hours, and 24 hours, DNA obtained from Lymphocytes 695 cells exhibits a significant peak having a size distribution commensurate with the expected size distribution of high molecular weight gDNA. Figure 5 Different treatment conditions to generate cfDNA-like DNA fragments affect the detection of aneuploid and euploid chromosomes in aneuploidy assays

Results of aneuploidy assay comparing chromosome ratios with euploid patient control samples and cfDNA-like DNA fragments generated from aneuploid cell line fibroblast 397 and euploid cell line Lymphocyte 695 cells across treatment conditions and post-treatment timepoints by dPCR. Each numbered panel depicts the chromosome ratios for the following conditions for each comparative chromosome pair assayed as indicated by the X axis labels. 1 : Euploidy patient control. 2: cfDNA-like DNA fragments from Fibroblast 397 cells at 4 hours post DMSO treatment. 3: cfDNA-like DNA fragments from Fibroblast 397 cells at 4 hours post control medium treatment. 4: cfDNA-like DNA fragments from Fibroblast 397 cells at 12 hours post control medium treatment. 5: cfDNA-like DNA fragments from Fibroblast 397 cells at 12 hours post monesin treatment. 6: cfDNA-like DNA fragments from Fibroblast 397 cells at 24 hours post monesin treatment. 7: cfDNA-like DNA fragments from Fibroblast 397 cells at 4 hours post monesin treatment. 8: cfDNA-like DNA fragments from Lymphocytes 695 cells at 4 hours post DMSO treatment. 9: cfDNA-like DNA fragments from Lymphocytes 695 cells at 12 hours post control medium treatment. 10: cfDNA-like DNA fragments from Lymphocytes 695 cells at 4 hours post control medium treatment. 11 : cfDNA-like DNA fragments from Lymphocytes 695 cells at 12 hours post monesin treatment. 12: cfDNA-like DNA fragments from Lymphocytes 695 cells at 4 hours post monesin treatment.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated 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 belongs, such as molecular genetics, cell culture, organic chemistry, and nucleic acid chemistry and hybridization.

Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology and genetics, cell culture, organic chemistry, and nucleic acid chemistry and hybridization, which are within the capabilities of a person of ordinary skill in the art. Standard techniques are used for nucleic acid synthesis.

The techniques and procedures are generally performed according to conventional methods in the art (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps and features referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only.

As used herein, the term "comprising" means any of the recited elements are necessarily included and other elements may optionally be included as well. "Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of” means that all elements other than those listed are excluded. 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 encompasses any or all possible combinations of one or more associated listed items.

The terms “a,” “an,” or “the” as used herein may not only include aspects with one member, but may also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a bead” may include a plurality of such beads and reference to “the sequence” may include reference to one or more sequences known to those skilled in the art, and so forth.

Terminology:

The term ‘deoxyribonucleotide base’ as used herein, typically contains a nucleobase, a deoxyribose sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases 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. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide. For the purposes of this invention, the terms ‘nucleotide’, ‘deoxyribonucleotide base’ and ‘base’ are used interchangeably. The term ‘DNA fragment’ as used herein, is a macromolecule comprising at least two or more covalently-linked deoxyribonucleotide bases in which the 3' and 5' ends on each base are joined by phosphodiester bonds. DNA fragments may be double stranded or single stranded, or comprise one or more double stranded regions and one or more single regions. DNA fragments may be linear or circular, and may be derived from DNA in linear or circular conformation, in either single- or double-stranded conformation, or more highly aggregated hybridization motifs. DNA and DNA fragments may comprise any combination of any nucleotides. One or more nucleotides in any DNA fragment of this disclosure may be modified, for example, comprising one or more chemical modifications of a nucleotide. Modified nucleotides may occur singularly or in a plurality with other nucleotide modifications that are the same modification or a different modification, which may be contiguous or non-contiguous with other nucleotide modifications that are the same modification or a different modification. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.

The terms ‘cell-free DNA’ or ‘cfDNA’ refer to extracellular DNA that has been extruded from cells, which may be typically obtained from a non-cellular fraction of a bodily fluid, such as blood, or a cell-free biological fluid, for example, whole blood processed to remove cells, urine, saliva, or other biological fluid. Typically, cfDNA for analysis is obtained from whole blood processed to remove cells, e.g., a plasma or serum sample. As used herein, ‘natural cfDNA’ or ‘naturally- occurring cfDNA’ refers to cfDNA as present in a biological sample, in contrast to genomic DNA fragments that are obtained by other methods (e.g., fragmentation, sonication).

Typically, cfDNA is approximately 150 to 200 bp in length, predominantly approximately 166 bp long, which, without wishing to be bound by theory, corresponds to the length of DNA wrapped around a nucleosome plus a linker. Without further wishing to be limited by theory, the length of cfDNA may result due to enzymatic cleavage in vivo from germline DNA, which typically results in fragments that are between 150 and 200 bp in length, and/or may be a product of cell apoptosis and cell breakdown, which provides basis for cfDNA often having a range of lengths across a spectrum. Consequently, cfDNA may be released from necrotic or apoptotic cells, and may be associated with apoptotic bodies, nucleosomes, or in another extracellular form.

The term ‘cfDNA-like DNA fragments’ refers to DNA fragments generated according to the methods disclosed herein, having properties similar to natural cfDNA which facilitate similar performance in an assay (e.g. NIPT) compared to natural cfDNA, such as cfDNA derived from a patient, e.g. a pregnant women or foetus. The term ‘NIPT’ refers to non-invasive prenatal testing, which includes methods to determine the risk for a foetus being born with a chromosomal abnormality, such as trisomy 21 , by analysing foetal cfDNA present in a sample, typically a blood sample, of a pregnant woman.

A ‘gene’, as used herein, is a segment of nucleic acid (typically DNA) that encodes the sequence information required for the production of a polypeptide or ribonucleic acid gene product. It includes regions preceding and following the coding region (5’ UTR and 3’UTR) as well as intervening sequences (introns) between individual coding segments (exons). Conveniently, this term also includes the necessary control sequences for gene expression (e.g. enhancers, silencers, promoters, terminators etc.), which may be adjacent to or distant to the relevant coding sequence, as well as the coding and/or transcribed regions encoding the gene product.

The term ‘copy number variation’ (CNV) refers to the variable number of copies of a specific segment of DNA between different individuals’ genomes or within an individual’s genome across different cell or tissue types. The variant regions are of variable length, typically defined as segments greater than 1 ,000 base pairs in length and less than 5 megabases in length. CNVs include both additional copies of a sequence as well as losses of genetic material, which may have come about through duplications, deletions, or other structural changes (e.g. by recombination). Such regions may or may not contain one or more genes. CNVs may be detected by qPCR or dPCR, wherein the copy number of a specific genomic region may manifest in the number of detected molecules or partitions which contain a target oligonucleotide originating from the specific genomic region.

The term ‘diagnostic assay’ as used herein, refers to any assay, screen, test, or method that may be used to characterize a genotype, such as aneuploidy, copy number variant, allelomorphism, polymorphism, splice variant, regulatory variant, mutation, indel, trinucleotide repeat, premature stop codon, translocation, somatic rearrangement, gene fusion, genetic alteration, or the presence of foreign or exogenous nucleotide sequences (e.g., a provirus), by analysing a cfDNA sample. For example, a diagnostic assay may refer to any type of nucleotide sequencing and subsequent analysis, or a diagnostic assay may comprise any type of nucleotide sequencing and subsequent analysis. A diagnostic assay may refer to quantitative PCR (qPCR) or digital PCR (dPCR), or a diagnostic assay may comprise qPCR or dPCR. A diagnostic assay may refer to next generation sequencing (‘NGS’) or a diagnostic test may comprise NGS, e.g., and subsequent analysis. A diagnostic assay may refer to nucleic acid hybridization, such as DNA microarray analysis. Similarly, a diagnostic assay may comprise nucleic acid hybridization, such as DNA microarray analysis. The term ‘polymerase chain reaction’ or ‘PCR’ as used herein refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

Reaction characteristics of a PCR reaction, such as amplification rate, or reaction outputs, such as a detectable number of molecules, may be detected and measured. These PCR methods may comprise a detection reagent or a detectable label which can be detected using any of a variety of detector devices. Exemplary detection methods include optical detection (e.g., fluorescence, or chemiluminescence) as well as others known to those of skill in the art. As a non-limiting example, a fluorescent label can be detected using a detector device equipped with a module to generate excitation light that can be absorbed by a fluorophore, as well as a module to detect light emitted by the fluorophore.

In embodiments, the diagnostic assay may comprise a digital PCR (dPCR) reaction, for example, a droplet digital PCR (ddPCR) reaction. Methods for performing PCR in partitions, such as droplets, are described, for example, in US 2014/0162266, US 2014/0302503, and US 2015/0031034, the contents of each of which is incorporated by reference. In embodiments, the QX200, QX600, or QX One Droplet Digital PCR (ddPCR) System (Bio-Rad) may be used. In embodiments, the diagnostic assay may comprise a qPCR reaction.

Methods for generating cfDNA-like DNA fragments

Disclosed herein are methods to prepare cfDNA-like DNA fragments which are suitable for a broad range of cell types and culturing applications. The disclosed methods of generating cfDNA-like DNA fragments comprise inducing apoptosis in a target cell line, resulting in the release of cfDNA-like DNA fragments into the surrounding media, which are subsequently extracted and size selected. cfDNA-like DNA fragments produced according to the methods of the disclosure accurately capture the size distribution and diagnostic assay performance, e.g. NIPT assays, of natural cfDNA, whilst beneficially producing a high yield and/or large quantities of cfDNA-like DNA fragments. Furthermore, the methods disclosed herein have many advantages, e.g., simple to perform, short production cycle, low-cost, suitable for a wide range of cell types, and can be readily and easily scaled up for large-scale production. The cfDNA-like DNA fragments prepared by the methods described herein can be widely used for assay methodological validation, internal assay quality control and external assay quality evaluation with high reproducibility and consistency. Exemplary assays include NIPT, which may include, but is not limited to: detection of aneuploidy, trisomy, CNV, and microdeletion(s). The cfDNA-like DNA fragments prepared by the methods described herein can be used as a reference in diagnostic assays in conjunction with non-invasive cfDNA samples, as well as invasive samples; e.g., from amniocentesis or CVS. cfDNA-like DNA fragments generated according to the methods may be suitable for patient-like controls or reference materials for in vitro screening, testing, and/or diagnostics utilising circulating cell free DNA (cfDNA) as a biomarker of interest, including, but not limited to: aneuploidy, trisomy, and microdeletion detections.

In aspects of the 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 cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments.

The methods and systems disclosed herein may be carried out using any suitable cell line obtained from or extracted from any animal. Typically, the target cell line is human in origin, but alternatively it may be from another animal, particularly a mammal, such as a mouse model organism, or from commercially farmed animals, such as horses, cattle, sheep, or pigs; or may alternatively be from pets such as cats or dogs.

The target cell line may be an adherent cell line, and/or may be a suspension cell line, or may be cultured under conditions wherein the target cell line has the characteristics of an adherent cell line or a suspension cell line. The target cell line may form 2 dimensional and/or 3 dimensional cellular structures, and/or may be cultured in 3D culture, and/or may comprise organoids. In embodiments, the target cell line is an adherent cell line. In alternative embodiments, the target cell line is a suspension cell line.

Suitable target cell lines are known to those skilled in the art, which include but are not limited to, Lymphocytes 695 (B-lymphocyte cell 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-21 H, MCF7, HTERT-HME1 , SiHa, SK-BR-3, T-47d, EFO-21 , BEWO, AN3-CA, HeLa, HUVEC TERT2, TIME, HSkMC, BJ hTERT+, BJ, ASC TERT1 , ASC diff, U- 2197,11-2 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.

The target cell line may be selected on the basis that a condition of interest is present, particularly wherein early detection of the condition (or associated disease) is desired. For example, the condition of interest may be a chromosomal abnormality of a human foetus (e.g. trisomy 21 , trisomy 18). Such suitable target cell lines include any cell line exhibiting a condition of interest, which may include but are not limited to: Fibroblast 397 (fibroblast cell line AG05397; Trisomy 21), Detroit 532 (Trisomy 21), UVWVC1-2DS3 (Down syndrome, Trisomy 21), UWWC1- DS1 (Down syndrome, Trisomy 21), UVWVC1-DS4 (Down syndrome, Trisomy 21), WC-24-02- DS-M (Down syndrome, Trisomy 21), WC-24-02-DS-O (Down syndrome, Trisomy 21), WC-24- 02-DS-P (Down syndrome, Trisomy 21). In embodiments, the target cell line is Lymphocytes 695 or Fibroblast 397.

Exemplary culture medium for use in the method may comprise, consist, or consist essentially of any culture medium routinely used for cell culture which is suitable for culturing the target cell line, for example, which include, but are not limited to: Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM),F-12K Medium, RPMI 1640 optionally comprising FBS, S-MEM (Serum-Free Medium), McCoy’s 5A Medium, Basal Medium Eagle (BME). In embodiments, the culturing medium may comprise Foetal bovine serum (FBS).

In aspects and embodiments, a medium comprising Dimethyl sulfoxide (DMSO) is employed to induce apoptosis in the target cell line, releasing cfDNA-like DNA fragments into the medium comprising DMSO. Culture systems are closed environments, typically in a culture dish or flask with cells surrounded by nutrient rich medium. It is in this medium that any nucleic acids released from the cells will accumulate and/or be slowly further degraded.

Experimental data disclosed herein demonstrates that inducing apoptosis with DMSO results in a higher cfDNA-like DNA fragment yield, and/or improved performance in NIPT aneuploidy assays compared to apoptosis inducing treatments known in the art.

In embodiments, the medium comprising DMSO comprises between about 40% and 1%, between about 35% and 5%, between about 30% and 10%, between about 25% and 15%, or between about 18% and 22% DMSO. In embodiments, the medium comprising DMSO comprises about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO. In embodiments, the medium comprising DMSO 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, e.g. about 20% DMSO.

In embodiments, the medium comprising DMSO comprises up to about 20% foetal 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 embodiments, the medium comprising DMSO comprises between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS. In embodiments, the medium comprising DMSO 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.

In embodiments, the medium comprising DMSO comprises about 40% DMSO and about 60% 5% FBS in culture medium, about 35% DMSO and about 65% 5% FBS in culture medium, about 30% DMSO and about 70% 5% FBS in culture medium, about 25% DMSO and about 75% 5% FBS in culture medium, about 20% DMSO and about 80% 5% FBS in culture medium, about 15% DMSO and about 85% 5% FBS in culture medium, about 10% DMSO and about 90% 5% FBS in culture medium, about 5% DMSO and about 95% 5% FBS in culture medium, or about 1% DMSO and about 99% 5% FBS in culture medium. In embodiments, the medium comprising DMSO comprises about 25% DMSO and about 75% 5% FBS in culture medium, about 24% DMSO and about 76% 5% FBS in culture medium, about 23% DMSO and about 77% 5% FBS in culture medium, about 22% DMSO and about 78% 5% FBS in culture medium, about 21% DMSO and about 79% 5% FBS in culture medium, about 20% DMSO and about 80% 5% FBS in culture medium, about 19% DMSO and about 81% 5% FBS in culture medium, about 18% DMSO and about 82% 5% FBS in culture medium, about 17% DMSO and about 83% 5% FBS in culture medium, about 16% DMSO and about 84% 5% FBS in culture medium, or about 15% DMSO and about 85% 5% FBS in culture medium.

In embodiments, the medium comprising DMSO comprises about 40% DMSO and about 60% culture medium comprising 5% FBS; about 35% DMSO and about 65% culture medium comprising 5% FBS; about 30% DMSO and about 70% culture medium comprising 5% FBS; about 25% DMSO and about 75% culture medium comprising 5% FBS; about 20% DMSO and about 80% culture medium comprising 5% FBS; about 15% DMSO and about 85% culture medium comprising 5% FBS; about 10% DMSO and about 90% culture medium comprising 5% FBS; about 5% DMSO and about 95% culture medium comprising 5% FBS; or about 1% DMSO and about 99% culture medium comprising 5% FBS. In embodiments, the medium comprising DMSO comprises about 25% DMSO and about 75% culture medium comprising 5% FBS; about 24% DMSO and about 76% culture medium comprising 5% FBS; about 23% DMSO and about 77% culture medium comprising 5% FBS; about 22% DMSO and about 78% culture medium comprising 5% FBS; about 21% DMSO and about 79% culture medium comprising 5% FBS; about 20% DMSO and about 80% culture medium comprising 5% FBS; about 19% DMSO and about 81% culture medium comprising 5% FBS; about 18% DMSO and about 82% culture medium comprising 5% FBS; about 17% DMSO and about 83% culture medium comprising 5% FBS; about 16% DMSO and about 84% culture medium comprising 5% FBS; or about 15% DMSO and about 85% culture medium comprising 5% FBS.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO to induce apoptosis. Experimental data disclosed herein demonstrates that the duration of incubation has an effect on the viability of the target cell line, which in turn, appears to have an effect on the quality of the cfDNA-like DNA fragments, e.g. a longer than optimal incubation time produces cfDNA fragments having a shorter than desirable fragment length distribution, whereas a shorter than optimal incubation time may reduce the yield of cfDNA-like DNA fragments. It is a benefit of the disclosed methods that a specific range of apoptosis induction is achieved, which without wishing to be bound by any particular theory, appears to result in an improved cfDNA-like DNA fragment yield and NIPT assay performance more similar to that of natural cfDNA.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours or up to about 4 hours. In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours. In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for about 0.5, about 1 , about 2, about 3, or about 4 hours. In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for between about 0.5 and about 12 hours, between about 1 and about 6 hours, between about 2 and about 4 hours, or between about 3 and about 4 hours. In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO at a temperature of about 37°C.

In aspects of the invention, a method for generating a plurality of cfDNA-like DNA fragments from a target adherent cell line is provided, the method comprising: i. removing a culturing medium from the target adherent cell line; ii. contacting cells of the target adherent cell line with a medium comprising DMSO; iii. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments. Optionally, the method may comprise first providing a target adherent cell line.

In aspects of the invention, a method for generating a plurality of cfDNA-like DNA fragments from a target suspension cell line is provided, the method comprising: i. separating cells of the target suspension cell line from a culturing medium; ii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments. Optionally, the method may comprise first providing a target adherent cell line.

In embodiments, separating cells of the target suspension cell line from a culturing medium comprises: separating a culturing medium comprising the target suspension cell line by sedimentation to obtain a culturing medium supernatant and a sedimented target suspension cell line; and removing the culturing medium supernatant from the sedimented target suspension cell line. In embodiments, the sedimented target suspension cell line may be produced by centrifugation or by gravity separation. In embodiments, separating cells of the target suspension cell line from a culturing medium may comprise removing the culturing medium supernatant from the sedimented target suspension cell line by aspiration.

In embodiments, contacting separated cells of the target suspension cell line with a medium comprising DMSO comprises resuspending sedimented cells of the target suspension cell line of cells in the medium comprising DMSO.

In embodiments, cells of the target cell line may be obtained by a cell sorting method, and the method comprises contacting the sorted cells with the medium comprising DMSO, e.g. sorting cells of the target cell line into the medium comprising DMSO.

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

In embodiments, incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments. In embodiments, the methods further comprise extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

In embodiments, cfDNA-like DNA fragments may be extracted from the medium comprising DMSO after incubating the medium comprising DMSO with the target cell line.

In embodiments, extracting cfDNA-like DNA fragments from the medium comprising DMSO comprises collecting the medium comprising a plurality of cfDNA-like DNA fragments and performing DNA extraction on the medium comprising a plurality of cfDNA-like DNA fragments to obtain a sample comprising a plurality of cfDNA-like DNA fragments.

In embodiments, extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments comprises: collecting the medium comprising DMSO and cfDNA-like DNA fragments; and performing DNA extraction on the medium comprising DMSO and cfDNA- like DNA fragments to obtain a sample comprising a plurality of cfDNA-like DNA fragments.

Numerous methods for extracting cfDNA from a liquid sample, e.g. medium, are known in the art. The general methods of DNA preparation (e.g., described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3d ed., 2001) can be followed. Extracting cfDNA from a liquid sample can be achieved e.g., by employing columns, beads, magnetic beads, or other isolation procedures. Kits for extracting cfDNA from samples are commercially available, such as QIAamp Circulating Nucleic Acid Kit (Qiagen), QiaAmp DNA Mini Kit (Qiagen), or Apostle Extraction kit (Beckman), may also be used to obtain cfDNA from sample. In embodiments, the DNA extraction method comprises the use of the QIAamp Circulating Nucleic Acid Kit (Qiagen).

In embodiments, 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%, about 99.9%.

It is a benefit of the invention that the DNA does not require extensive cleanup, e.g. removal from exosomes, and therefore may benefit from one or more of improved speed, cost, and simplicity.

In embodiments, the cfDNA-like DNA fragments have a desired length. In embodiments, the method comprises obtaining cfDNA-like DNA fragments having a desired length. In embodiments, the method further comprises obtaining cfDNA-like DNA fragments having a desired length. cfDN A-like DNA fragments can be processed by subjecting the cfDNA-like DNA fragments to a method that generates a distribution of cfDNA-like DNA fragments having a specific distribution of fragment length or having a desired length.

In embodiments, obtaining cfDNA-like DNA fragments having a desired length comprises performing size selection on the cfDNA-like DNA fragments of the sample comprising a plurality of cfDNA-like DNA fragments.

In embodiments, obtaining cfDNA-like DNA fragments having a desired length may comprise performing size selection on the cfDNA-like DNA fragments.

Methods for performing DNA size selection are well known in the art and encompass a variety of techniques to isolate DNA fragments having a distribution of desired lengths. Methods employed in the art include, but are not limited to: gel electrophoretic separation of DNA fragments, excision, and purification of excised electrophoresis gel based on a target size distribution; restriction enzyme digestion to cleave DNA at specific recognition sites, yielding DNA fragments of predetermined sizes; size exclusion chromatography; and bead-based size selection methods.

Methods of DNA size selection typically employ magnetic or paramagnetic, including superparamagnetic, or glass, coated beads e.g. coated with a polymer, which selectively bind and isolate DNA fragments of a specific size range, depending on the concentration of beads and the ratio of DNA to beads used in the reaction.

Commonly used beads include, but are not limited to: silica-coated magnetic beads, carboxylate-modified magnetic beads, amine-blocked magnetic beads, oligo(dT)-coated magnetic beads, streptavidin-coated magnetic beads, streptavidin-blocked magnetic beads, mag-sepharose, and Glassmilk beads. Methods of creating beads suitable for DNA size selection are known in the art, (see: https://www.protocols.io/view/home-brew-spri-beads- eq2ly3mkmgx9/v1) or alternatively, beads for performing DNA size selection are commercially available, such as SPRIselect (Beckman) or AMPure XP Beads (Beckman).

After incubation of a DNA sample with magnetic beads for DNA size selection, the DNA-bound beads may be captured using a magnetic stand, allowing the supernatant comprising either undesired DNA fragments to be removed and discarded, or for the supernatant comprising desired DNA fragments to be obtained, optionally for subsequent size selection steps, e.g. a double-sided size selection comprising an additional bead incubation. Subsequent washing steps further purify the DNA-bound beads, ensuring the retention of only the desired fragment sizes. The purified, retained DNA fragments may then be eluted from the beads. In embodiments, performing size selection on the cfDNA-like DNA fragments comprises the use of AMPure XP Beads (Beckman).

In embodiments, the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally, about 160 bp. In embodiments, the desirable length of the 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.

In embodiments, the desirable length of the cfDNA-like DNA fragments is about 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 embodiments, the desirable length of the cfDNA-like DNA fragments is about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 bp. In embodiments, the desirable length of the cfDNA-like DNA fragments is about 150, 155, 160, 165, or 170 bp. In embodiments, the desirable length of the cfDNA-like DNA fragments is less than about 500, 400, 300, 200, or 170 bp.

It is a benefit of the invention that the cfDNA-like DNA fragments generated by the disclosed methods accurately capture characteristics of natural cfDNA. Experimental data disclosed herein demonstrates that cfDNA-like DNA fragments generated by the disclosed methods have a desirably narrower size distribution of cfDNA-like DNA fragments compared to methods currently employed in the art, including those which comprise apoptosis induction (for example, see Figure 3). Furthermore, as demonstrated by electrophoretic data filed herewith, the DNA fragment size of each peak and the percentage of each peak representing the cfDNA in the extracted and size selected sample closely matches that of natural cfDNA.

The inventors are the first to discover that treating a target cell line with DMSO, suitably a specific concentration of DMSO, results in the production of DNA fragments which are an improved facsimile of cfDNA obtained from patients compared to methods of the art, such as US11753682B2, evidenced at least by their size distribution and performance in diagnostic assays such as aneuploidy assays, which closely approximates I ‘mimics’ that of cfDNA obtained from patient samples.

In particular, the inventors are the first to demonstrate that the use of DMSO specifically as a chemical for inducing apoptosis generates cfDNA-like DNA fragments having the desirable properties of cfDNA obtained from patient samples, in contrast to other well-known methods or chemicals used for inducing apoptosis, e.g. monesin and cisplatin. Furthermore, the inventors are the first to demonstrate that the incubation time of DMSO, suitably a medium comprising DMSO, with a target cell line may substantially affects the properties of the cfDNA-like DNA fragments, and also their performance in diagnostic assays such as aneuploidy assays.

Notably, the methods of the invention produce cfDNA-like DNA fragments which have improved consistency in their performance in aneuploidy assays, e.g. aneuploid chromosomes and euploid chromosomes are correctly detected in all cell lines tested, in contrast to methods known in the art and other treatment conditions which induce apoptosis, and which may even produce DNA fragments having a desirable size distribution, but which do not perform as well as the cfDNA-like DNA fragments generated according to the methods of the invention.

As such, in embodiments, dPCR or qPCR is performed on the cfDNA-like DNA fragments obtained according to the methods of the invention to obtain a relative abundance of a first chromosome compared to a second chromosome. in embodiments, dPCR or qPCR is performed on the cfDNA-like DNA fragments obtained according to the methods of the invention to obtain a relative abundance of a first genomic region compared to a second genomic region, e.g. to obtain a copy number of a first genomic region and a copy number of a second genomic region.

In embodiments, the relative abundance of a first chromosome compared to a second chromosome comprises a first chromosome ratio between the first chromosome and the second chromosome.

In embodiments, the relative abundance of a first chromosome compared to a second chromosome, or the relative abundance of a first genomic region compared to a 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; suitably, wherein the relative abundance of a first chromosome compared to a second chromosome comprises a first chromosome ratio.

In embodiments, 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. In embodiments, 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; suitably, wherein the target cell line is a target adherent cell line or a target suspension cell line.

In embodiments, the first chromosome ratio is between about 1 .40 to 1 .60, between about 1 .42 to 1 .58, between about 1 .44 to 1 .56, between about 1 .46 to 1 .54, or between about 1 .48 to 1 .52, e.g. about 1 .5.

In embodiments, 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, suitably wherein the target cell line is a target adherent cell line or a target suspension cell line.

In embodiments, 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.

In embodiments, the first chromosome ratio is between about 0.9 to 1.1 , between about 0.92 to 1 .08, between about 0.94 to 1 .06, between about 0.96 to 1 .04, between about 0.98 to 1 .02, e.g. about 1.0.

In embodiments, the first chromosome ratio of the cfDNA-like DNA fragments is similar to a chromosome ratio obtained from a patient cfDNA sample.

In embodiments, the first ratio of the cfDNA-like DNA fragments is similar to a ratio obtained from a patient cfDNA sample; suitably, wherein the first chromosome ratio of the cfDNA-like DNA fragments is similar to a chromosome ratio obtained from a patient cfDNA sample.

In embodiments, the first chromosome ratio of the cfDNA-like DNA fragments may be up to about 20% different to a chromosome ratio obtained from a patient cfDNA sample, such as 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% different to a chromosome ratio from a patient cfDNA sample, e.g. 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%.

In embodiments, the first ratio of the cfDNA-like DNA fragments is up to about 20% different to a ratio obtained from a patient cfDNA sample, such as 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% different to a chromosome ratio from a patient cfDNA sample, e.g. 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 wherein the first ratio is a first chromosome ratio.

As such, the cfDNA-like DNA fragments generated according to the methods of the invention may be particularly beneficial for use as NIPT assay reference material, and in the development of NIPT assays, which may comprise dPCR or qPCR assays.

The methods disclosed herein beneficially constitute an optimal balance of high cfDNA-like DNA fragment yield with cfDNA-like DNA fragments having NIPT assay performance similar to that of natural cfDNA, which without wishing to be bound by any particular theory, appears to be achieved by a finely tuned range of apoptosis induction. Without wishing to be bound by theory, it is believed that the specific induction of apoptosis is a further benefit of the invention, since there are many types of cell death, of which apoptosis is only one pathway. Indeed, the induction of cell death alone appears to be insufficient to achieve the desired cfDNA-like DNA fragments, as demonstrated in Figures 1 , 2 and 5, wherein increased cell death does not correlate with both an increased yield and a desirable profile of cfDNA-like DNA fragments.

Provision of cfDNA-like DNA fragments

Disclosed herein are cfDNA-like DNA fragments which are suitable for a broad range of cell types and culturing applications. The disclosed cfDNA-like DNA fragments are obtained by inducing apoptosis in a target cell line, resulting in the release of cfDNA-like DNA fragments into the surrounding media, which may be subsequently extracted and size selected. The provided cfDNA-like DNA fragments accurately capture the size distribution and diagnostic assay performance, e.g. NIPT assays, of natural cfDNA, and are beneficially able to be produced at a high yield and/or in large quantities. The production of the disclosed cfDNA-like DNA fragments is beneficially simple to perform, has a short production cycle, is low-cost, and is suitable for a wide range of cell types, which can be readily and easily scaled up for large-scale production.

The cfDNA-like DNA fragments described herein can be widely used for assay methodological validation, internal assay quality control and external assay quality evaluation with high reproducibility and consistency. Exemplary assays include NIPT, which may include, but is not limited to: detection of aneuploidy, trisomy, CNV, and microdeletion(s). The cfDNA-like DNA fragments described herein can be used as a reference in diagnostic assays in conjunction with non-invasive cfDNA samples, as well as invasive samples; e.g., from amniocentesis or CVS. cfDNA-like DNA fragments described herein may be suitable for patient-like controls or reference materials for in vitro screening, testing, and/or diagnostics utilising circulating cell free DNA (cfDNA) as a biomarker of interest, including, but not limited to: aneuploidy, trisomy, and microdeletion detections.

In general, the methods of the invention comprise: the steps of: contacting a plurality of target cells with a medium comprising DMSO, and incubating the cells with the medium comprising DMSO to induce apoptosis; thereby to generate a plurality of cfDNA-like DNA fragments. The cells may be from a target cell line. The methods of the invention may comprising providing a target cell line and contacting cells thereof with the medium comprising DMSO.

In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate the plurality of cfDNA-like DNA fragments

In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: i. providing a target cell line ii. lysing cells of the target cell line with a buffer comprising ATAC-RSB to obtain DNA of the target cell line; and iii. digesting the DNA of the target cell line with Mnase to generate cfDNA-like DNA fragments.

In embodiments, the target cell line is an adherent cell line.

In embodiments, the target cell line is a suspension cell line.

In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: 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 medium comprising DMSO; iv. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments. In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: i. providing a target adherent cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target adherent cell line; iii. incubating the cells of the target adherent cell line with the culture medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: i. providing a target suspension cell line; ii. separating cells of the target suspension cell line from a culturing medium; iii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iv. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In aspects of the invention, a plurality of cfDNA-like DNA fragments is provided, produced by the process of: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target suspension cell line; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

In embodiments, separating cells of the target suspension cell line from a culturing medium comprises: separating a culturing medium comprising the target suspension cell line by sedimentation to obtain a culturing medium supernatant and a sedimented target suspension cell line; and removing the culturing medium supernatant from the sedimented target suspension cell line.

In embodiments, the sedimented target suspension cell line is produced by centrifugation or by gravity separation. In embodiments, separating cells of the target suspension cell line from a culturing medium comprises removing the culturing medium supernatant from the sedimented target suspension cell line by aspiration.

In embodiments, incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

In embodiments, the process further comprises extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

In embodiments, extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments comprises: collecting the medium comprising DMSO and cfDNA-like DNA fragments; and performing DNA extraction on the medium comprising DMSO and cfDNA-like DNA fragments to obtain a sample comprising a plurality of cfDNA-like DNA fragments.

In embodiments, the cfDNA-like DNA fragments have a desired length.

In embodiments, the process further comprises performing size selection on the cfDNA-like DNA fragments of the sample comprising a plurality of cfDNA-like DNA fragments to obtain cfDNA-like DNA fragments having a desired length.

In embodiments, size selection comprises mono-nucleosomal size selection.

In embodiments, the desirable length of the 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.

In embodiments, the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally, about 160 bp. In embodiments, the medium comprising DMSO comprises between about 40% and 1%, between about 35% and 5%, between about 30% and 10%, or between about 25% and 15% DMSO.

In embodiments, the medium comprising DMSO comprises about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1 % DMSO.

In embodiments, the medium comprising DMSO comprises 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 embodiments, the medium comprising DMSO comprises between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS.

In embodiments, the medium comprising DMSO 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.

In embodiments, the medium comprising DMSO comprises about 40% DMSO and about 60% 5% FBS in culture medium, about 35% DMSO and about 65% 5% FBS in culture medium, about 30% DMSO and about 70% 5% FBS in culture medium, about 25% DMSO and about 75% 5% FBS in culture medium, about 20% DMSO and about 80% 5% FBS in culture medium, about 15% DMSO and about 85% 5% FBS in culture medium, about 10% DMSO and about 90% 5% FBS in culture medium, about 5% DMSO and about 95% 5% FBS in culture medium, or about 1% DMSO and about 99% 5% FBS in culture medium.

In embodiments, the medium comprising DMSO comprises about 25% DMSO and about 75% 5% FBS in culture medium, about 24% DMSO and about 76% 5% FBS in culture medium, about 23% DMSO and about 77% 5% FBS in culture medium, about 22% DMSO and about 78% 5% FBS in culture medium, about 21 % DMSO and about 79% 5% FBS in culture medium, about 20% DMSO and about 80% 5% FBS in culture medium, about 19% DMSO and about 81% 5% FBS in culture medium, about 18% DMSO and about 82% 5% FBS in culture medium, about 17% DMSO and about 83% 5% FBS in culture medium, about 16% DMSO and about 84% 5% FBS in culture medium, or about 15% DMSO and about 85% 5% FBS in culture medium.

In embodiments, the medium comprising DMSO comprises about 40% DMSO and about 60% culture medium comprising 5% FBS, about 35% DMSO and about 65% culture medium comprising 5% FBS, about 30% DMSO and about 70% culture medium comprising 5% FBS, about 25% DMSO and about 75% culture medium comprising 5% FBS, about 20% DMSO and about 80% culture medium comprising 5% FBS, about 15% DMSO and about 85% culture medium comprising 5% FBS, about 10% DMSO and about 90% culture medium comprising 5% FBS, about 5% DMSO and about 95% culture medium comprising 5% FBS, or about 1% DMSO and about 99% culture medium comprising 5% FBS.

In embodiments, the medium comprising DMSO comprises about 25% DMSO and about 75% culture medium comprising 5% FBS, about 24% DMSO and about 76% culture medium comprising 5% FBS, about 23% DMSO and about 77% culture medium comprising 5% FBS, about 22% DMSO and about 78% culture medium comprising 5% FBS, about 21% DMSO and about 79% culture medium comprising 5% FBS, about 20% DMSO and about 80% culture medium comprising 5% FBS, about 19% DMSO and about 81% culture medium comprising 5% FBS, about 18% DMSO and about 82% culture medium comprising 5% FBS, about 17% DMSO and about 83% culture medium comprising 5% FBS, about 16% DMSO and about 84% culture medium comprising 5% FBS, or about 15% DMSO and about 85% culture medium comprising 5% FBS.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours or up to about 4 hours.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO for about 0.5, about 1 , about 2, about 3, or about 4 hours.

In embodiments, the cells of the target cell line are incubated with the medium comprising DMSO at a temperature of about 37°C. In embodiments, comprising assaying copy number variability of a region of DNA in the target cell line based on the presence of cfDNA-like DNA fragments.

In embodiments, comprising performing a dPCR reaction or a qPCR reaction.

In embodiments, dPCR or qPCR is performed on the cfDNA-like DNA fragments to assay the foetal fraction of a biological sample and/or copy number variability of a region of DNA.

In embodiments, dPCR or qPCR is performed on the cfDNA-like DNA fragments to obtain a relative abundance of a first chromosome compared to a second chromosome.

In embodiments, the relative abundance of a first chromosome compared to a second chromosome, or the relative abundance of a first genomic region compared to a 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, suitably wherein the relative abundance of a first chromosome compared to a second chromosome comprises a first chromosome ratio.

In embodiments, 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.

In embodiments, 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, suitably wherein target cell line is an target adherent cell line or target suspension cell line.

In embodiments, 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, suitably wherein target cell line is a target adherent cell line or target suspension cell line. In embodiments, 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.

In embodiments, the first chromosome ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a chromosome ratio obtained from a patient cfDNA sample.

In embodiments, the first ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a ratio obtained from a patient cfDNA sample, suitably wherein the first chromosome ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a chromosome ratio obtained from a patient cfDNA sample.

In embodiments, the first chromosome ratio of the cfDNA-like DNA fragments is about 25% different to a chromosome ratio obtained from a patient cfDNA sample, such as about 20%, about 15%, about 12.5%, about 10%, about 7.5%, about 5%, about 2.5%, or about 1% different to a chromosome ratio obtained from a patient cfDNA sample, e.g. 10%.

In embodiments, the first ratio of the cfDNA-like DNA fragments is up to about 20% different to a ratio obtained from a patient cfDNA sample, such as 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% different to a chromosome ratio from a patient cfDNA sample, e.g. 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 a first chromosome ratio.

EXAMPLES

The invention will now be further illustrated by way of the following non-limiting examples. Unless otherwise indicated, standard techniques in molecular biology, cell culture, biochemistry, and nucleic acid chemistry were used.

Materials and Methods Examples of the following procedures used in accordance with this disclosure are described below:

Apoptosis induction and release of cfDNA fragments in an adherent target cell line

The target adherent cell line was cultured in an appropriate serum-containing cell culture medium in 3 to 4 150 cm² flasks to a confluency of 90 to 100%. Prior to DMSO treatment, the culture medium was aspirated from each flask and replaced with 16 mL of a 5% FBS culture medium comprising DMSO in a ratio of 1 :5 of DMSO to medium in each flask. The target adherent cell line was incubated with the medium comprising DMSO for 4 hours in a standard cell culture incubator under standard conditions (37°C, 5% CO2, and 18.6% to 20.9% O2). After incubation, the supernatant/medium comprising DMSO was collected in 50 mL tubes, and either stored at -70°C to -80°C, or processed by DNA extraction.

Apoptosis induction and release of cfDNA fragments in a suspension target cell line

The target suspension cell line was cultured in an appropriate serum-containing cell culture medium in 2 to 3 150 cm² flasks to a confluency of 90 to 100%. Prior to DMSO treatment, each cell culture was transferred to a separate 50 mL conical tube, and centrifuged at 500 RCF (x g) for 5 minutes to from a pellet of cells. The culture medium was aspirated from each conical tube and replaced with 16 mL of a 5% FBS culture medium comprising DMSO in a ratio of 1 :5 of DMSO to medium in each conical tube. The pellet of cells was subsequently resuspended in the medium comprising DMSO. The target suspension cell line was incubated with the medium comprising DMSO for 4 hours in a standard cell culture incubator under standard conditions (37°C, 5% CO2, and 18.6% to 20.9% O2). After incubation, each conical flask comprising the target suspension cell line was centrifuged at 500 RCF (x g) for 5 minutes to from a pellet of cells, and the supernatant/medium comprising DMSO was collected in 50 mL tubes, and either stored at -70°C to -80°C, or processed by cfDNA extraction. cfDNA extraction cfDNA extraction was performed using the QIAamp Circulating Nucleic Acid kit (Qiagen, Cat # 55114) according to manufacturer’s instructions. cfDNA collection based on the method of US11753682B2

Briefly, cells of the selected cell line are treated with ionophore monensin at a concentration of 5 to 15 pM at 37°C in a humidified atmosphere of 5% CO2 for at least 1 day (24 hours). The culture media is then centrifuged at 200g for 30 mins at RT. Supernatant is removed and mixed with an equal volume of the total exosome isolation reagent. The cell culture media is incubated at 4°C overnight. After incubation, the media is centrifuged at 10,000g for 1 h at 4°C. The supernatant is discarded and the pellet resuspended in PBS and stored at -80°C. cfDNA can be isolated from the extracellular vesicle pellet with a silica-based membrane for binding, such as QIAGEN circulating nuclei acid isolation kit.

Size selection

0.8x + 1.5x Size selection (for obtaining short fragments and fragments having a desirable length)

Prior to commencing size selection of cfDNA, AM Pure XP beads were equilibrated at room temperature for 15 min, and then vortexed for several seconds. A 0.8x volume relative to the original extracted cfDNA sample volume of AM Pure XP was added to the extracted cfDNA and mixed by briefly vortexing the cfDNA-bead mixture. The cfDNA-bead mixture was then incubated for 15 minutes at room temperature, and subsequently transferred to a magnet for a further 5 to 10 minute incubation until the mixture became clear and the beads had collected at one side/corner of the vessel containing the mixture. The clear supernatant was then transferred to a new vessel, and the original vessel containing the beads was retained for QC of bound fragments. A 1.5x volume relative to the original extracted cfDNA sample volume of AMPure XP was added to the clear supernatant in the new vessel, and mixed by either briefly vortexing or pipetting the cfDNA-bead mixture. The cfDNA-bead mixture was then incubated for 5 minutes at room temperature, and subsequently transferred to a magnet for a further 5 to 10 minute incubation until the mixture became clear and the beads had collected at one side/corner of the vessel containing the mixture. The clear supernatant was then removed. A 2x volume of 70% ethanol was added to all vessels containing beads whilst in contact with a magnet, including the original vessel containing the first beads, and incubated for 30 seconds at room temperature. The 70% ethanol was then removed from the vessels whilst in contact with a magnet. A further 2x volume of 70% ethanol was added to all vessels containing beads whilst in contact with a magnet, and incubated for 30 seconds at room temperature. The 70% ethanol was then removed from the vessels whilst in contact with a magnet, and the beads left to air dry for 5 to 10 minutes. The vessels containing beads were then removed from the magnet and a 1.0x volume relative to the original extracted cfDNA sample volume of elution buffer was added to all vessels containing beads, and mixed by pipetting. The vessels containing the elution bufferbead mixture were then incubated for 5 minutes at room temperature, and subsequently transferred to a magnet for a further 5 to 10 minute incubation until the elution buffer-bead mixture became clear and the beads had collected at one side/corner of the vessel containing the elution buffer-bead mixture. The clear supernatant was then transferred to a new vessel.

Fragment size analysis

The fragment length distribution of the cfDNA-like DNA fragments obtained by the method was analysed using a High-Sensitivity DNA Bioanalyzer kit (Agilent) on the Bioanalyzer platform (Agilent). ddPCR

The chromosomal ratios of the cfDNA-like DNA fragments were analysed by performing a dPCR aneuploidy assay using 1.5 to 10 ng of the cfDNA-like DNA fragments. The uniformity of target representation was assessed by calculating the ratios between target concentrations. cdDNA- like DNA fragments having ratios within about ± 30%, within about ± 25%, within about ± 20%, within about ± 15%, or within about ± 10%, e.g. within about ± 20% of the expected ratio are considered to accurately reflect natural cfDNA distributions and were used for further QC. Any available method for performing a dPCR aneuploidy assay may be used by those skilled in the art to evaluate the cfDNA-like DNA fragments.

The following examples further illustrate aspects and embodiments of the methods of the present disclosure.

Example 1

As can be seen in Figure 1 A, at 4 hours post treatment for the Fibroblast 397 cell line, cisplatin treatment yields the lowest percentage of viable cells at approximately 28%, followed by monesin at approximately 41 %, and DMSO at approximately 52%. At 12 hours post-treatment, the percentage of viable cells for the DMSO and monesin conditions, at approximately 23% and 31% respectively, is substantially lower than at 4 hours post-treatment. In contrast, the percentage of viable cells for the cisplatin condition, at approximately 26%, is generally comparable to the viability of the cells treated with cisplatin at 4 hours post-treatment. At 24 hours post-treatment, the cell viability for each condition is generally comparable to the cell viability at 12 hours post-treatment.

For the Lymphocyte 695 cell line, as can be seen in Figure 1 B, at 4 hours post treatment, cisplatin treatment yields the lowest percentage of viable cells at approximately 3%, followed by DMSO at approximately 24%, and monsin at approximately 39%. At 12 hours post-treatment, the percentage of viable cells for the DMSO and monesin conditions, at approximately 17% and 30% respectively, is substantially lower than at 4 hours post-treatment. In contrast, the percentage of viable cells for the cisplatin condition, is approximately 9%. At 24 hours posttreatment, DMSO treatment yields the lowest percentage of viable cells at approximately 7%, followed by cisplatin at approximately 12%, and monesin at approximately 38%.

Example 2 Figure 2 demonstrates comparatively increased total DNA yield from DMSO treated Fibroblast 397 cells compared to treatment with monesin, cisplatin, or control media. Notably, comparatively less total DNA is obtained from Fibroblast 397 cells 4 hours post DMSO treatment than at 12 hours and 24 hours.

In contrast, Figure 2 demonstrates that the media treatment conditions produced a comparatively (substantially) greater yield of DNA compared to all other conditions at all posttreatment timepoints tested for the Lymphocyte 695 cell line. Furthermore, at all post-treatment timepoints tested, the monesin treatment condition produced a comparatively (substantially) greater yield of DNA compared to the DMSO treatment conditions for the Lymphocyte 695 cell line.

Example 3

Figure 3A and 3B demonstrates that the cfDNA-like DNA fragments produced according to the methods of the invention (apoptosis method) accurately capture the size distribution of patient cfDNA, having the expected average length of ~170 bp and similar distribution around ~170 bp.

Furthermore, as can be seen in Figure 3B, the proportion of the sample I DNA fragments comprising cfDNA-DNA like fragments having a size distribution similar to that of patient cfDNA, is either comparable to cfDNA-like material generation methods utilising monesin as described in US11753682B2, referred to in Figure 3B as ‘BT’, or higher than comparable methods for generating cfDNA-like material.

Additionally, as can be seen in Figure 3B, compared to methods comprising the use of monesin, as described in US11753682B2, the cfDNA-like DNA fragments obtained at 4 hours post DMSO treatment according to the methods of the invention have a narrower distribution of cfDNA-like DNA fragments, which is more similar to the patient samples than the method of US11753682B2, which exhibits a comparatively wider distribution of fragments than the patient samples and the DMSO apoptosis method.

Example 4

Figure 4 demonstrates that a comparatively higher amount of total DNA does not equate to a higher amount of cfDNA-like DNA fragments having the desirable size distribution similar to patient cfDNA. For example, whilst the 4 hours post-media treatment condition for Lymphocyte 695 cells produces a higher yield of total DNA than DMSO as can be seen in Figure 2B, the total DNA comprises a substantially greater amount of HMW DNA compared to the desirable cfDNA-like DNA fragments as can be seen in Figure 4C. Additionally, whilst the 4 hours post-monensin treatment condition for Lymphocyte 695 cells produces a higher yield of total DNA as can be seen in Figure 2B, as can be seen in Figure 4C, this DNA is almost entirely HMW DNA, which is not desirable.

As evidenced in Figure 4B, the 4 hours post DMSO treatment condition yields cfDNA having a size distribution commensurate with cfDNA obtained from a patient, and also yields little to no HMW fragments consistently across the cell types tested. Notably, the 4 hours post DMSO treatment condition yields the most similar cfDNA-like DNA fragment distribution to the patient sample.

Notably, the cfDNA-like DNA fragments of which the data in Figure 4 relates have not been size selected, and thus represent cfDNA-like DNA fragments obtained just from extraction of cfDNA- like fragments from medium post-treatment.

Example 5

Figure 5 demonstrates that the conditions which exhibit the chromosome ratios closest to all of the expected chromosome ratios for euploid and aneuploid samples is the 4 hours post DMSO treatment condition, which exhibits ratios of aneuploid Chr21 versus all euploid chromosomes of approximately 1.5 (see lane 2 - aneuploid Fibroblast 397 cell line), and a ratio of euploid chromosomes to euploid chromosomes of 1.0 (see lane 2 - aneuploid Fibroblast 397 cell line, and also lane 8 - euploid Lymphocyte 695 cell line). Furthermore, the 4 hours post DMSO treatment condition exhibits decreased chromosome ratio variance at 4 hours in aneuploid cells compared to all other conditions.

In contrast, the monesin treatment condition at all timepoints for aneuploid Fibroblast 397 (trisomy 21) does not exhibit the expected chromosome 21/22q ratio of 1.5, producing inaccurate I misleading chromosome ratios.

Notably, the 4 hour and 12 hour media conditions do not produce desirable aneuploidy ratios, particularly the ratio of approximately 1.75 for the chromosome ratio of Chr21/Chr22q and Chr21/Chr13, is too high and, therefore, disadvantageous. In this regard, it would be misleading to use a control sample having a chromosomal ratio of greater than 1 .5 to create a contrived sample mimicking a 4% fetal fraction sample, as it would produce a higher chromosomal ratio than a real sample. If used to test the analytical power of an assay, for example, it would give unrealistically good results and overestimate the detection power of the assay. Furthermore, Figure 5 demonstrates that the DNA fragments generated under these conditions, whilst having a desirable size distribution, do not perform similarly to patient samples in aneuploidy assays or produce the expected chromosome ratios, indicating that the size distribution alone is not sufficient to recapture the properties of patient cfDNA samples or to generate the expected chromosome ratios.

In contrast, the 4 hours post DMSO treatment condition produces chromosome ratios that are expected for the aneuploid I euploid conditions of the two cell lines, e.g. for the 395 cell line (Chr21 aneuploid), chromosome ratio values of approx. 1.5 are obtained for Chr21/22q, Chr21/13, and Chr21/18, and a chromosome ratio value of approx. 1 is obtained for Chr18/13; and for the 696 cell line (euploid), all chromosome ratios are around 1.0, as expected.

Clauses

Expressions of the inventive concept are set out in the following clauses:

Clause group M (method clauses):

M1. A method for generating a plurality of cfDNA-like DNA fragments, the method comprising: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments.

M2. The method according to Clause M1 , wherein the target cell line is an adherent cell line.

M3. The method according to Clause M1 , wherein the target cell line is a suspension cell line.

M4. A method for generating a plurality of 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 medium comprising DMSO; iv. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

M5. A method for generating a plurality of cfDNA-like DNA fragments from a target adherent cell line, the method comprising: i. providing a target adherent cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target adherent cell line; iii. incubating the cells of the target adherent cell line with the culture medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

M6. A method for generating a plurality of cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing a target suspension cell line; ii. separating cells of the target suspension cell line from a culturing medium; iii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iv. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

M6a. A method for generating a plurality of cfDNA-like DNA fragments from a target suspension cell line, the method comprising: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target suspension cell line; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

M7. The method according to Clause M6, wherein separating cells of the target suspension cell line from a culturing medium comprises: separating a culturing medium comprising the target suspension cell line by sedimentation to obtain a culturing medium supernatant and a sedimented target suspension cell line; and removing the culturing medium supernatant from the sedimented target suspension cell line. M8. The method according to Clause M7, wherein the sedimented target suspension cell line is produced by centrifugation or by gravity separation.

M9. The method according to any of Clauses M6, M7 or M8, wherein separating cells of the target suspension cell line from a culturing medium comprises removing the culturing medium supernatant from the sedimented target suspension cell line by aspiration.

M10. The method according to any of Clauses M6, M7, M8 or M9, wherein contacting separated cells of the target suspension cell line with a medium comprising DMSO comprises resuspending sedimented cells of the target suspension cell line of cells in the medium comprising DMSO.

M11 . The method according to any preceding clause, wherein cells of the target cell line are obtained by a cell sorting method, and the method comprises contacting the sorted cells with the medium comprising DMSO.

M11a. The method according to any of the preceding clauses, wherein incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

M12. The method according to any preceding clause, further comprising extracting cfDNA- like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

M13. The method according to Clause M12, wherein extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments comprises: collecting the medium comprising DMSO and cfDNA-like DNA fragments; and performing DNA extraction on the medium comprising DMSO and cfDNA-like DNA fragments to obtain a sample comprising a plurality of cfDNA-like DNA fragments.

M14. The method of any preceding clause, comprising obtaining cfDNA-like DNA fragments having a desired length.

M14a. The method of any preceding clause, wherein the cfDNA-like DNA fragments have a desired length. M15. The method according to Clause M14, wherein obtaining cfDNA-like DNA fragments having a desired length comprises performing size selection on the cfDNA-like DNA fragments of the sample comprising a plurality of cfDNA-like DNA fragments.

M16. The method according to Clause M15, wherein size selection comprises mono- nucleosomal size selection.

M17. The method according to any of Clauses M14 to M16, wherein the desirable length of the 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.

M18. The method according to any of Clauses M14 to M17, wherein the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally, about 160 bp.

M19. The method according to any preceding clause, wherein the medium comprising DMSO comprises between about 40% and 1%, between about 35% and 5%, between about 30% and 10%, or between about 25% and 15% DMSO.

M20. The method according to any preceding clause, wherein the medium comprising DMSO comprises about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO.

M21 . The method according to any preceding clause, wherein the medium comprising DMSO 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.

M22. The method according to any preceding clause, wherein the medium comprising DMSO comprises 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.

M23. The method according to any preceding clause, wherein the medium comprising DMSO comprises between about 0.5% and 20% FBS, between about 1 % and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS. M24. The method according to any preceding clause, wherein the medium comprising DMSO 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.

M25. The method according to any preceding clause, wherein the medium comprising DMSO comprises about 40% DMSO and about 60% culture medium comprising 5% FBS, about 35% DMSO and about 65% culture medium comprising 5% FBS, about 30% DMSO and about 70% culture medium comprising 5% FBS, about 25% DMSO and about 75% culture medium comprising 5% FBS, about 20% DMSO and about 80% culture medium comprising 5% FBS, about 15% DMSO and about 85% culture medium comprising 5% FBS, about 10% DMSO and about 90% culture medium comprising 5% FBS, about 5% DMSO and about 95% culture medium comprising 5% FBS, or about 1% DMSO and about 99% culture medium comprising 5% FBS.

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

M27. The method according to any preceding clause, wherein the cells of the target cell line are incubated with the medium comprising DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours or up to about 4 hours.

M28. The method according to any preceding clause, wherein the cells of the target cell line are incubated with the medium comprising DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

M29. The method according to any preceding clause, wherein the cells of the target cell line are incubated with the medium comprising DMSO for about 0.5, about 1 , about 2, about 3, or about 4 hours. M29a. The method according to any preceding clause, wherein the cells of the target cell line are incubated with the medium comprising DMSO for between about 0.5 and 8 hours, between about 0.5 and 7 hours, between about 1 and 7 hours, between about 1 and 6 hours, between about 2 and 6 hours, between about 2 and 5 hours, between about 2.5 and 5 hours, between about 2.5 and 4.5 hours, between about 3 and 4.5 hours, or between about 3 and 4 hours.

M30. The method according to any preceding clause, wherein the cells of the target cell line are incubated with the medium comprising DMSO at a temperature of about 37°C.

M31. The method according to any preceding clause, comprising assaying copy number variability of a region of DNA in the target cell line based on the presence of cfDNA-like DNA fragments.

M32. The method according to any preceding clause, comprising performing a dPCR reaction or a qPCR reaction.

M33. The method according to Clause M32, wherein dPCR or qPCR is performed on the cfDNA-like DNA fragments to assay the foetal fraction of a biological sample.

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

M35. The method according to Clause M34, wherein the relative abundance of a first chromosome compared to a second chromosome, or the relative abundance of a first genomic region compared to a 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, suitably wherein the relative abundance of a first chromosome compared to a second chromosome comprises a first chromosome ratio.

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.

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 target suspension cell line.

M38. The method according to any one of clauses M35, or M36 or M37 when dependent on Clause M35, wherein the first chromosome ratio is between about 1.40 to 1.60, between about 1.42 to 1.58, between about 1.44 to 1.56, between about 1.46 to 1.54, or between about 1.48 to 1.52, e.g. about 1.5.

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 and the second chromosome is known or expected to be euploid in the target cell line, suitably wherein the target cell line is a target adherent cell line or target suspension cell line.

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.

M41 . The method according to any one of clauses M35, or M39 or M40 when dependent on Clause M35, wherein the first chromosome ratio is between about 0.9 to 1.1 , between about 0.92 to 1.08, between about 0.94 to 1.06, between about 0.96 to 1.04, between about 0.98 to 1.02, e.g. about 1.0.

M42. The method according to any one of clauses M35, or M36 to M41 when dependent on Clause M35, wherein the first ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a ratio obtained from a patient cfDNA sample, suitably wherein the first chromosome ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a chromosome ratio obtained from a patient cfDNA sample.

M43. The method according to any one of clauses M35, or M36 to M41 when dependent on Clause M35, wherein the first ratio of the cfDNA-like DNA fragments is up to about 15% different to a ratio obtained from a patient cfDNA sample, such as 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% different to a chromosome ratio from a patient cfDNA sample.

M43a. The method according to any one of clauses M35, or M36 to M41 when dependent on Clause M35, wherein the first ratio of the cfDNA-like DNA fragments is between about -10% and +10% different to a ratio obtained from a patient cfDNA sample, such as between about - 8% and +8%, between about -6% and +6%, between about -4% and +4% or between about - 2% and +2% different to a chromosome ratio from a patient cfDNA sample.

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

M44. A method for generating a plurality of cfDNA-like DNA fragments, the method comprising: i. providing a target cell line ii. lysing cells of the target cell line with a buffer comprising ATAC-RSB to obtain DNA of the target cell line; and iii. digesting the DNA of the target cell line with Mnase to generate cfDNA-like DNA fragments.

Clause group P (product clauses):

P1. A plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate the plurality of cfDNA-like DNA fragments.

P2. The plurality of cfDNA-like DNA fragments according to Clause P1 , wherein the target cell line is an adherent cell line.

P3. The plurality of cfDNA-like DNA fragments according to Clause P1 , wherein the target cell line is a suspension cell line.

P4. A plurality of cfDNA-like DNA fragments, produced by the process of: 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 medium comprising DMSO; iv. incubating the cells of the target adherent cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

P5. A plurality of cfDNA-like DNA fragments, produced by the process of: iv. providing a target adherent cell line in a culture medium; v. adding DMSO to the culture medium contacting the target adherent cell line; vi. incubating the cells of the target adherent cell line with the culture medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

P6. A plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target suspension cell line; ii. separating cells of the target suspension cell line from a culturing medium; iii. contacting separated cells of the target suspension cell line with a medium comprising DMSO; iv. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

P6a. A plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target suspension cell line in a culture medium; ii. adding DMSO to the culture medium contacting the target suspension cell line; iii. incubating the separated cells of the target suspension cell line with the medium comprising DMSO to induce apoptosis and generate cfDNA-like DNA fragments.

P7. The plurality of cfDNA-like DNA fragments according to Clause P6, wherein separating cells of the target suspension cell line from a culturing medium comprises: separating a culturing medium comprising the target suspension cell line by sedimentation to obtain a culturing medium supernatant and a sedimented target suspension cell line; and removing the culturing medium supernatant from the sedimented target suspension cell line.

P8. The plurality of cfDNA-like DNA fragments according to Clause P7, wherein the sedimented target suspension cell line is produced by centrifugation or by gravity separation.

P9. The plurality of cfDNA-like DNA fragments according to any of Clauses P6, P7 or P8, wherein separating cells of the target suspension cell line from a culturing medium comprises removing the culturing medium supernatant from the sedimented target suspension cell line by aspiration. P10. The plurality of cfDNA-like DNA fragments according to any of Clauses P6, P7, P8 or P9, wherein contacting separated cells of the target suspension cell line with a medium comprising DMSO comprises resuspending sedimented cells of the target suspension cell line of cells in the medium comprising DMSO.

P11. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P10, wherein incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

P12. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P11 , the process further comprising extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

P13. The plurality of cfDNA-like DNA fragments according to Clause P12, wherein extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments comprises: collecting the medium comprising DMSO and cfDNA-like DNA fragments; and performing DNA extraction on the medium comprising DMSO and cfDNA-like DNA fragments to obtain a sample comprising a plurality of cfDNA-like DNA fragments.

P14. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P13, wherein the cfDNA-like DNA fragments have a desired length.

P15. The plurality of cfDNA-like DNA fragments according to Clause P14, the process further comprising performing size selection on the cfDNA-like DNA fragments of the sample comprising a plurality of cfDNA-like DNA fragments to obtain cfDNA-like DNA fragments having a desired length.

P16. The plurality of cfDNA-like DNA fragments according to Clause P15, wherein size selection comprises mono-nucleosomal size selection.

P17. The plurality of cfDNA-like DNA fragments according to any of Clauses P14 to P16, wherein the desirable length of the 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. P18. The plurality of cfDNA-like DNA fragments according to any of Clauses P14 to P17, wherein the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally about 160 bp.

P19. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P18, wherein the medium comprising DMSO comprises between about 40% and 1%, between about 35% and 5%, between about 30% and 10%, or between about 25% and 15% DMSO.

P20. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P19, wherein the medium comprising DMSO comprises about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% DMSO.

P21. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P20, wherein the medium comprising DMSO 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.

P22. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P21 , wherein the medium comprising DMSO comprises 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.

P23. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P22, wherein the medium comprising DMSO comprises between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS.

P24. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P23, wherein the medium comprising DMSO 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.

P25. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P24, wherein the medium comprising DMSO comprises about 40% DMSO and about 60% culture medium comprising 5% FBS, about 35% DMSO and about 65% culture medium comprising 5% FBS, about 30% DMSO and about 70% culture medium comprising 5% FBS, about 25% DMSO and about 75% culture medium comprising 5% FBS, about 20% DMSO and about 80% culture medium comprising 5% FBS, about 15% DMSO and about 85% culture medium comprising 5% FBS, about 10% DMSO and about 90% culture medium comprising 5% FBS, about 5% DMSO and about 95% culture medium comprising 5% FBS, or about 1 % DMSO and about 99% culture medium comprising 5% FBS.

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

P27. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P26, wherein the cells of the target cell line are incubated with the medium comprising DMSO for up to about 24 hours, up to about 12 hours, up to about 6 hours or up to about 4 hours.

P28. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P27, wherein the cells of the target cell line are incubated with the medium comprising DMSO for at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or at least about 4 hours.

P29. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P28, wherein the cells of the target cell line are incubated with the medium comprising DMSO for about 0.5, about 1 , about 2, about 3, or about 4 hours.

P30. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P29, wherein the cells of the target cell line are incubated with the medium comprising DMSO for between about 0.5 and 8 hours, between about 0.5 and 7 hours, between about 1 and 7 hours, between about 1 and 6 hours, between about 2 and 6 hours, between about 2 and 5 hours, between about 2.5 and 5 hours, between about 2.5 and 4.5 hours, between about 3 and 4.5 hours, or between about 3 and 4 hours.

P31. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P30, wherein the cells of the target cell line are incubated with the medium comprising DMSO at a temperature of about 37°C. P32. The plurality of cfDNA-like DNA fragments according to any of Clauses P1 to P31 , comprising assaying copy number variability of a region of DNA in the target cell line based on the presence of cfDNA-like DNA fragments.

P33. A plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target cell line ii. lysing cells of the target cell line with a buffer comprising ATAC-RSB to obtain DNA of the target cell line; and iii. digesting the DNA of the target cell line with Mnase to generate cfDNA-like DNA fragments.

Claims

CLAIMS:

1. A method for generating a plurality of cfDNA-like DNA fragments, the method comprising: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments.

2. The method according to Claim 1 , wherein incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

3. The method according to Claim 2, further comprising extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

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

5. The method according to Claim 4, wherein the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally, about 160 bp.

6. The method according to any one of Claims 1 , 2, or 3, wherein the medium comprising DMSO 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 cells of the target cell line are incubated with the medium comprising DMSO for between about 0.5 and 8 hours, between about 0.5 and 7 hours, between about 1 and 7 hours, between about 1 and 6 hours, between about 2 and 6 hours, between about 2 and 5 hours, between about 2.5 and 5 hours, between about 2.5 and 4.5 hours, between about 3 and 4.5 hours, or between about 3 and 4 hours.

8. The method according to any one of the Claims 1 , 2, or 3, wherein the medium comprising DMSO comprises between about 0.5% and 20% FBS, between about 1% and 10% FBS, between about 2% and 9% FBS, between about 3% and 8% FBS, between about 4% and 7% FBS, or between about 5% and 6% FBS.

9. The method according to Claim 1 , wherein dPCR or qPCR is performed on the cfDNA- like DNA fragments to obtain a relative abundance of a first chromosome compared to a second chromosome ora first genomic region compared to a second genomic region; optionally wherein the relative abundance of a first chromosome compared to a second chromosome, or the relative abundance of a first genomic region compared to a second genomic region, comprises a first chromosome ratio between the first chromosome and the second chromosome; optionally, wherein the first chromosome ratio of the cfDNA-like DNA fragments is identical to, substantially the same as, or similar to a chromosome ratio obtained from a patient cfDNA sample.

10. The method according to 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 according to Claim 9 or Claim 10, wherein the first chromosome ratio of the cfDNA-like DNA fragments is up to about 15% different to a chromosome ratio obtained from a patient cfDNA sample, such as 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 % different to a chromosome ratio from a patient cfDNA sample, e.g. up to about 10%.

12. The method according to Claim 9 or Claim 10, wherein the first chromosome ratio is between about 1 .40 to 1 .60, between about 1 .42 to 1 .58, between about 1 .44 to 1 .56, between about 1 .46 to 1 .54, or between about 1 .48 to 1 .52, e.g. about 1 .5.

13. A plurality of cfDNA-like DNA fragments, produced by the process of: i. providing a target cell line; ii. contacting cells of the target cell line with a medium comprising DMSO; and iii. incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate the plurality of cfDNA-like DNA fragments.

14. The plurality of cfDNA-like DNA fragments according to Claim 13, wherein incubating the cells of the target cell line with the medium comprising DMSO to induce apoptosis of cells of the target cell line and generate cfDNA-like DNA fragments releases cfDNA-like DNA fragments into the medium comprising DMSO, generating a medium comprising DMSO and cfDNA-like DNA fragments.

15. The plurality of cfDNA-like DNA fragments according to Claim 13 or Claim 14, the process further comprising extracting cfDNA-like DNA fragments from the medium comprising DMSO and cfDNA-like DNA fragments.

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

17. The plurality of cfDNA-like DNA fragments according to Claim 13 or Claim 14, wherein the desirable length of the cfDNA-like DNA fragments is between about 150 bp and 170 bp; optionally, about 160 bp.

18. The plurality of cfDNA-like DNA fragments according to Claim 13 or Claim 14, wherein the medium comprising DMSO 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.

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

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