Reference material comprising nucleic acids having a fragmentation and nucleosome profile and diagnostic method

Abstract

The present invention relates to a novel reference material comprising nucleic acids having a specific fragmentation pattern, in particular for use in sequencing methods such as polymerase chain reaction (PCR) or next generation sequencing (NGS), process for production and use thereof and to an associated diagnostic method. Said reference material allows the validation of such methods including the verification of measurement results, especially according to instrument parameters and sample preparation. The invention further relates to an associated diagnostic method relating to a liquid biopsy or cell-free DNA.

Description

[0001] Reference material containing nucleic acids with a fragmentation and nucleosome profile and diagnostics

[0002] Description

[0003] The present invention relates to a novel reference material comprising nucleic acids with a specific fragmentation pattern, in particular for use in sequencing methods such as polymerase chain reaction (PCR) or next-generation sequencing (NGS), methods for its production and use, and associated diagnostics. This reference material allows the validation of such methods, including the verification of measurement results, in particular as a function of device parameters and sample preparation. Furthermore, the invention encompasses an associated diagnostic method for a liquid biopsy or cell-free DNA.

[0004] Despite all the progress made in recent years, current methods for detecting diseases, such as clinical chemical tests in body fluids and tissue samples or imaging techniques, have often led to the late detection of adverse changes. In order to prevent the occurrence of metastases in tumor diseases, for example, it is necessary to detect malignant changes as early as possible. Furthermore, methods are needed to correctly distinguish diseased cells from healthy cells. Both false-positive and false-negative findings have fatal consequences for those affected. Likewise, methods are required that allow a correct prognosis and a correspondingly tailored treatment for the individual patient.

[0005] Allow therapy. The isolation, characterization, and analysis of nucleic acids (ribonucleic acids, RNA and deoxyribonucleic acids, DNA) from cell materials is usually performed in the following steps in the current state of the art: sample preparation, extraction, concentration, isolation, purification, if necessary, reverse transcription, amplification, and detection.

[0006] Due to the powerful amplification of DNA by polymerase chain reaction (PCR), as used in "qPCR" (real-time quantitative polymerase chain reaction), "digital PCR" (dPCR), or various variants of "next generation sequencing" (NGS, or parallel sequencing such as "massive parallel sequencing"), the so-called minimally to non-invasive liquid biopsy has become established in diagnostics, in which, for example, circulating free DNA in a sample (blood, plasma) is examined. Such circulating free DNA (cfDNA for short) are short double- or single-stranded DNA molecules (50-200 bp) from the fragmentation of genomic DNA from apoptotic and necrotic cells, and are used as biomarkers for the diagnosis of diseases.

[0007] In cancer patients and other diseases, the average amount of cfDNA is higher than in healthy subjects (so-called ctDNA in tumor diseases). In particular, the cfDNA plasma level is higher in advanced cancer patients than in mild / early stage cancer patients. For example, ctDNA accounts for 1% of cfDNA (0.001-90% of normal cfDNA, depending on the stage and location of the tumor).

[0008] It is further known that cfDNA originating from various cell types, e.g., from various healthy organs / tissues or pathologically altered organs / tissues, e.g., tumors at different anatomical sites, circulating fetal cfDNA (of primary placental origin) and tumor ctDNA are shorter than "background" cfDNA, which is primarily of hematopoietic origin. cfDNA also bears signatures of its association with nucleosomes and chromatosomes, such as the most abundant plasma DNA length of 166 bp and fragmentation endpoints that show relationships to nucleosome arrangement.

[0009] A nucleosome is the unit of DNA and a histone octamer. The octamer consists of two copies each of the proteins H2A, H2B, H3 and H4. 146 or 147 base pairs of DNA are wound around such a protein complex in a left-handed superhelix. The chromatosome results from the binding of histone H1 to a nucleosome. The chromatosome contains 166 base pairs of DNA. In the following, however, they are referred to collectively as nucleosome(s). In all eukaryotes, DNA exists in a condensed state called chromatin.

[0010] While cfDNA spanning the entire genome (from both the nuclear and mitochondrial genomes) is present in plasma, there are microheterogeneities in cfDNA coverage between different genomic regions. For example, cfDNA abundance and fragment end frequencies differ between open and closed chromatin domains, regions within and between nucleosomes, and specific genetic elements such as transcription factor binding sites. There is evidence that cfDNA has a predisposition to be cleaved at certain genomic regions or elements, thereby exhibiting a higher representation of certain end sequence motifs. Such selectivity of cleavage sites may be linked to the sequence motif preferences of the nucleases involved in the process. These relationships are the subject of so-called "fragmentomics."

[0011] Chromatin structure influences the length and frequency of cfDNA. Open (euchromatin) and closed chromatin regions (heterochromatin) differ in the frequency of nucleosomes and the level of gene expression (e.g., through the presence of transcription factor (TF) binding sites), which in turn can increase or decrease accessibility to nucleases. These variables are manifested in the fragmentomic features of a cfDNA. The majority of cfDNA is mononucleosomal in length, and the nucleosomes influence the length of the cfDNA (nucleosome footprint).

[0012] Understanding the fragmentation patterns of cfDNA depending on the cell type can aid in the identification of pathologies / diseases associated with these tissues or organs. Furthermore, it has recently been shown that fragmentation patterns can differ depending on the tissue of origin (i.e., fetal or maternal, cancer or non-cancerous) or nucleosomal packaging of the DNA. Therefore, the fragmentation pattern includes not only the fragment sizes but also the fragment endpoints and nucleosome footprints. Furthermore, gene expression, i.e., the number of copies or RNA transcripts, is affected.

[0013] However, the major challenges of fragmentomics include preanalytical aspects such as blood collection and the timing of blood transport to the laboratory and processing, particularly sample preparation. Furthermore, certain DNA extraction and library preparation protocols can preferentially deplete or enrich DNA fragments of certain sizes, so these steps must be carefully planned for meaningful DNA fragmentation analyses. Biological aspects include, for example, the various nucleases involved in fragmentation (e.g., DNA fragmentation factor beta, DNASE1L3, and DNASEI), which sometimes leave behind overhanging ends with a defined sequence or base background, as well as different sizes.

[0014] For these reasons, the cf DNA coverage or sequence depth (so-called "coverage") is not uniform, but specific, depending on the cell type and nucleosome footprint, so that specific fragmentation patterns are obtained.

[0015] Coverage for DNA, specifically cfDNA, refers to the number of sequencing reads used to analyze the cfDNA. Coverage is typically defined as the average number of reads generated across a given genome or target region. High coverage indicates a sufficient number of reads to provide accurate information about the underlying DNA or cfDNA sequence. Low coverage, on the other hand, may result in inaccurate or missing information.

[0016] Adequate coverage is important for identifying rare genetic variants, mutations, or structural alterations in DNA or cfDNA. It also enables precise quantification of specific DNA sequences or analysis of cell-free DNA fragments in general.

[0017] The exact coverage requirement may vary depending on the type of analysis, the research question, and the sequencing technology used.

[0018] There is therefore a high demand for a suitable standard or reference material to enable validation or calibration of a cf DNA determination depending on the coverage requirement of a sequencing method, or related amplicon or non-amplicon methods such as polymerase chain reaction (PCR) or next-generation sequencing (NGS). Furthermore, instrument parameters are important.

[0019] Common laboratory standards are described in the state of the art.

[0020] A disadvantage, however, is that these internal laboratory standards have to be produced and diluted for the purpose of validating or calibrating a device or method, and there is no automated process available for this, so standardization is usually prone to errors. As a result, the copy number determined by sequencing, cycling, or non-amplicon-based methods can be relatively or absolutely incorrect due to a lack of sufficient standardization. Furthermore, cfDNA is usually not used, as this would have to be obtained from blood samples, and consequently there may be insufficient sequences available for analysis. The (blood) donors can also be sick or healthy and can therefore influence and falsify the standard. It is therefore necessary to produce artificial cfDNA as reference material, which has not yet been described in the state of the art.

[0021] Therefore, it is necessary to further improve standardization with the help of an artificial reference material that corresponds to natural cfDNA, has a natural fragmentation pattern (supra) and meets the coverage requirement.

[0022] The object of the present invention is therefore to provide an improved method for validating a sequencing method for the determination of natural cfDNA.

[0023] Surprisingly, the inventors were able to determine that, compared to such a standard, the use of cfDNA with a specific fragmentation pattern significantly improves validation. This requires considering the aforementioned coverage, so that an improved validation result can be achieved depending on the cell type, organ, or tissue. Furthermore, sufficient analyzable sequences must be available.

[0024] The problem is therefore solved by a method for validating a sequencing method for the determination of cfDNA, wherein i . ) a reference material is provided which is produced using the following steps: a . ) providing a cell culture comprising cells, b . ) treating the cell culture from a . ) with a cell division inhibitor, c.) isolating the nucleic acids comprising nucleosomes, d.) treating the nucleic acids comprising nucleosomes with nucleases and / or ultrasound, e.) isolating nucleic acids with a length of 20-750 bp, and ii.) the nucleic acids from e.) are determined by means of the sequencing method.

[0025] The above method is hereinafter referred to as the "validation method according to the invention".

[0026] Particularly advantageously, step b.) can be used to maintain or influence the coverage or the desired fragmentation pattern. The cell division inhibitor freezes the state or a selected coverage.

[0027] The advantage of the method according to the invention is that the reference material can be produced with as consistent a quality as possible across all production cycles. This is particularly difficult because each cell in that cell culture is at a different cell cycle stage. As a result, one cell will not have the same DNA content as another at the moment of its harvest. Likewise, the distribution of heterochromatin and euchromatin varies between the cells, affecting the transcriptome and the methylation pattern. To counteract this, the cells can be treated with a cell division inhibitor according to the invention, the aim of which is to transfer all cells into the same cell cycle at the time of harvest.

[0028] To advantageously preserve the entire DNA packed on nucleosomes in cell culture, the cells are preferably treated with a mitosis inhibitor, preferably colchicine (also called colchicine), so that mitosis is arrested in metaphase. This is advantageous because it ensures the highest possible coverage. Furthermore, it advantageously achieves consistent production of the reference material across each batch, as the cells can always be harvested in the same cell cycle. When colchicine is added to cultured eukaryotic cells, the cells enter mitosis and arrest in metaphase with condensed chromosomes.

[0029] Therefore, within the meaning of this invention, such inhibitors of cell division, in particular mitosis inhibitors or checkpoint inhibitors, can be used, in particular selected from the group Colcemide (CAS number: 477-30-5), Benomyl (CAS number: 17804-35-2), Nocodazole (CAS number: 31430-18-9), Paclitaxel (CAS number: 33069-62-4), Vincristine (CAS number: 57-22-7), Vinblastine (CAS number: 865-21-4), 8-Chloroadenosine (CAS number: 34408-14-5), WYE 687 dihydrochloride (CAS number: 1702364-87-1), AZD 5438 (CAS number: 602306-29-6) , Pladienolide B (CAS number: 445493-23-2) , Temsirolimus (CAS number: 162635-04-3) , Tosyl-L-Arginine Methyl Ester (TAME) (CAS number: 1784-03-8) , YC 1 (CAS number: 170632-47-0) , Indisulam (CAS number: 165668-41-7) .

[0030] Furthermore, nucleases according to the invention are used according to step d.), selected from the group MNase (EC number:

[0031] 3.1.31.1) , DNaselL3 (EC number: 3.1.21.1) , DNase I (EC number:

[0032] 3.1.21.1) .

[0033] Following enzymatic digestion, DNA can be purified and optionally end-repaired using Klenow / T4 DNA polymerase (EC number: 2.7.7.7). Alternatively, ultrasound can be used according to step d. ), allowing nucleic acids with a length of 20-750 bp to be obtained.

[0034] However, the use of nucleases according to step d ) is preferred.

[0035] For the purposes of this invention, "reference material" means a nucleic acid produced in steps a . ) - e . ) with a length of 20-750 bp, in particular 50-500 bp, in particular 20-200 bp . These produced nucleic acids are "artificial cfDNA" for the purposes of this invention or are cfDNA or represent cfDNA or a totality of cfDNA.

[0036] In a further embodiment of the invention, the cell culture comprising cells according to step a.) can have one or more specific cell types, in particular those capable of mitotic or meiotic cell division, selected from the group of human cells, mammalian cells, eukaryotic cells, somatic cells, stem cells, B cells, T cells, natural killer cells, diseased or healthy cells, tumor cells, cells from a tissue or organ. Also included are monoclonal cell cultures of immortal (immortal) eukaryotic cells, in particular those which have arisen from polyclonal cultures by isolation. Immortalized cells can arise through infection with viruses, e.g. infection of lymphocytes with Epstein Barr virus, or through the cultivation of tumor cells which have been removed from a diseased mammal. In this case, a mixed culture of many individual cells is initially obtained (so-called polyclonal culture).Using isolation techniques, new cell cultures can be created from individual cells (so-called monoclonal cultures). This isolation eliminates differences that can occur at the DNA level between cells of a polyclonal culture. This makes the material particularly clean and can be used, in particular, for the validation of highly sensitive methods. Such monoclonal cell cultures or immortalized cells are therefore particularly preferred due to the identity and homogeneity of the cells. Consequently, the reference material obtained according to the invention has few impurities.

[0037] The method according to the invention advantageously allows the provision of a reference material which represents the typical and specific fragmentation pattern.

[0038] For the purposes of this invention, "sequencing method" means any suitable sequencing method for sequencing nucleic acids, DNA, in particular cfDNA, in particular according to the Sanger method or so-called 2nd generation or amplicon or non-amplicon methods. Amplicon or non-amplicon methods are those such as pyrosequencing, sequencing by hybridization, ion semiconductor DNA sequencing system, nanopore sequencing, next generation sequencing (NGS), sequencing-by-synthesis (SBS), polymerase chain reaction (PCR), qPCR (real-time quantitative polymerase chain reaction), digital PCR (dPCR).

[0039] For the purposes of this invention, amplicon methods mean those that require an amplicon for sequencing. Here are some common sequencing methods used for amplicons: i. ) Sanger sequencing: This classic sequencing method is based on the use of terminating nucleotides to determine the DNA sequence. Sanger sequencing is often used for the analysis of short amplicons, typically up to a few hundred base pairs. ii. ) Next-generation sequencing (NGS): NGS technologies such as Illumina sequencing or Ion Torrent sequencing allow the sequencing of many amplicons simultaneously. With NGS, hundreds of thousands to millions of reads can be generated, resulting in a high throughput rate. NGS is suitable for sequencing amplicons of various lengths, from a few hundred to several thousand base pairs. iii. ) PacBio sequencing : The single-molecule

[0040] Sequencing technology from Pacific Biosciences (PacBio) enables the sequencing of long amplicons. It offers a very long read length and is therefore well suited for sequencing amplicons that span several thousand to ten thousand base pairs. iv.) Oxford Nanopore sequencing: The Oxford Nanopore

[0041] Sequencing also allows the sequencing of long amplicons. The technology is based on real-time sequencing by passing DNA molecules through a nanopore. It offers long read lengths and is portable.

[0042] For the purposes of this invention, non-amplicon methods mean sequencing methods that do not require amplicons.

[0043] For the purposes of this invention, validation means that, based on a validation sample comprising the nucleic acids (or reference material) produced according to the method a.) to e.) according to the invention, these can be determined by sequencing. This positive detection may depend on the instrument parameters on the one hand, and on the execution of the sequencing method on the other. The validation sample can be provided by means of a kit.

[0044] If the validation sample is not found during the sequencing procedure, the sensitivity is not given or the detection limit is too high, so that detection cannot be carried out.

[0045] A further particular embodiment of the invention relates to a method for diagnosing or prognosticating a disease, wherein cfDNA is determined, in particular for assessing the degree of severity and for monitoring the course of therapy, for investigating minimal residual disease (MRD) and for making therapy decisions, wherein calibration is carried out using the validation method according to the invention.

[0046] The method according to the invention particularly advantageously allows the detection limit of cfDNA to be validated. A particularly advantageous application therefore concerns the validation of devices for carrying out, in particular, a polymerase chain reaction (PCR) or next-generation sequencing (NGS), in which the method according to the invention is carried out.

[0047] Particularly advantageously, a precisely specified amount of DNA material or concentration of the reference material according to the invention can be introduced into a sample or comparison sample in this way, whereby this sample largely simulates a sample and is highly suitable for carrying out a diagnosis. The term "diagnosis" encompasses medical diagnostics and related examinations, in particular in vitro diagnostics and laboratory diagnostics. Preference is given to a statement about a disease or a condition of a patient, which can be obtained from the determination of cf DNA.

[0048] In the context of this invention, "patient" is defined as any

[0049] Subject understood.

[0050] The invention is explained below using examples and figures. However, the invention is not limited to the examples and figures, but is fundamentally universally applicable.

[0051] Example 1: Validation of a diagnostic procedure for the detection of minimal residual disease (MRD) or monitoring / follow up examinations in cancer.

[0052] In one study, fragmented DNA was used in an NGS procedure to validate it. For comparison, various other materials of non-natural origin were used to attempt to validate the system. Isolated cell-free DNA from a healthy donor was used as a normal control. It was shown that DNA prepared as described could be used to validate the system, whereas other materials were unsuitable due to their differences from the patient material.

[0053] In particular, the fragment size distribution and

[0054] Conversion efficiency was investigated. Under the

[0055] Conversion efficiency is understood as the successful processing of DNA fragments to prepare them for sequencing.

[0056] In order for the validation of a system, especially for the detection of MRD, to be carried out successfully, the material used for validation must be very similar to the patient material and particularly clean (have a low error rate).

[0057] It was shown that both the conversion efficiency and the size distribution of the material, when produced according to the invention, are so similar to the patient material that a validation of the system can be carried out successfully.

[0058] However, all other materials tested in the same experiment did not behave in the same way as the patient material.

[0059] Figure 1 :

[0060] A) The mean error rate (MER) was determined for various cfDNA-like control materials (RM) and cfDNA from healthy donors (black bar, HD cfDNA) using the MRD assay (supra). The samples were sequenced at comparable target depths of ~100,000x.

[0061] B ) The cfDNA produced in the method described here (see line for BDXXP4 n-cfDNA) has a size distribution that is most similar to the healthy patient cfDNA (black solid line, Healthy donor (HD) cfDNA).

[0062] Example 2: Validation of a diagnostic method for detecting a genetic change during a pregnancy test. The technologies mentioned, such as PCR and NGS methods, can also be used to detect diseases of an unborn child in the mother's blood. This can be done by a liquid biopsy and subsequent DNA extraction from the blood. The extracted DNA often contains a proportion of fetal DNA in addition to the maternal DNA. The proportion of fetal DNA is often in the range 1-10%. Fetal DNA differs from maternal DNA, among other things, in its size. On average, fetal DNA is smaller than maternal DNA. The present invention can therefore be used to validate technology that examines DNA extracted from mothers. This is done, for example, by...two DNA fragments of different lengths are mixed together in a ratio that is similar to the natural ratio.

[0063] In this example, an experiment was conducted to investigate whether a mixture of two DNA fragments of different lengths (see Figure 2B X2 and X3) could be distinguished from one another using a PCR method. It was found that the individual DNAs in the mixture (see Figure 2B X4) could be distinguished from one another using PCR methods.

[0064] Figure 2 :

[0065] Figure 2 shows:

[0066] (A) Possible differences in nucleosome density upon treatment with one or no cytotoxin;

[0067] (B) Possible fragmentation profiles after treatment with techniques that lead to randomized DNA fragments (XI, e.g., ultrasound), or treatment of the DNA with nucleases, which lead to cfDNA fragmentation profiles, such as those of healthy individuals (X2) and diseased individuals (X3). X4 = superposition of X2 and X3.

[0068] *Other cytotoxins can produce a different nucleosome density. This influences the coverage profile after treatment with a corresponding nuclease.

[0069] Figure 3:

[0070] Figure 3 shows (XI) the normal fragment length distribution in humans and (X2) overlay of DNA produced by the method according to the invention.

[0071] Figure 4 :

[0072] Possible methods for producing fragmented DNA.

[0073] DNA can be produced either by (1,2) nuclease digestion of DNA from cells treated with a cytotoxin, or (3) by nuclease digestion of DNA from cells not treated with a cytotoxin, or by (4) methods that result in randomized fragments after gDNA extraction. The method as shown in (1,2) can be carried out with cytotoxins that lead to arrest at different cell cycle phases. If cytotoxins that arrest the cell cycle in a phase that leads to a high chromatin density are used, as in the method shown in (1), this leads to high coverage. If cytotoxins that arrest the cell cycle in a phase that leads to a low chromatin density are used, as in the method shown in (2), this leads to low coverage.(1) and (2) have the advantage that the same coverage is achieved between different manufacturing batches with respect to defined sequences, while this is not achieved by methods as described in (3).

[0074] Consequently, better reproducibility in the

[0075] Production through the inventive use of cytotoxin, thus achieving a defined coverage. The particular advantage of (1) in the figure is that a higher and more uniform coverage is achieved across the entire sequence examined. By using colchicine, for example, most sequences are wrapped around histones (maximally condensed). This results in very high coverage across the entire genome.

[0076] Figure 5:

[0077] Figure 5 shows a coverage comparison between

[0078] Cell cycle inhibitor (+cytotoxin) treated and non-cell cycle inhibitor treated (-cytotoxin) cells after DNA fragmentation by different pathways.

[0079] (A) Coverage ratio between the individual preparation methods in the whole-genome overview. Whole exome sequencing (WES) data were collected. The comparison shows differences in coverage between DNA from cells treated with a cytotoxin that leads to high chromatin density (+ cytotoxin) and without cytotoxin.

[0080] (- cytotoxin) and subsequently digested with Mnase. In comparison, coverage is highest with randomly fragmented DNA.

[0081] (B) Detailed comparison of chromosomes 1 and 2. [XI] compares the coverage between non-cytotoxic

[0082] (- cytotoxin + nuclease) treated cells whose gDNA was randomly fragmented with nuclease-digested DNA from cells not treated with cytotoxin. The coverage is lower in cells not treated with cytotoxin, represented here by the downward deviation from the baseline. [X2] compares the coverage between non-cytotoxin-treated cells whose gDNA was randomly fragmented with nuclease-digested DNA from cells treated with cytotoxin.

[0083] (+ cytotoxin + nuclease). The coverage is lower in cells that were not treated with cytotoxin, shown here by the downward deviation from the baseline. [X3] compares the coverage between cells not treated with cytotoxin whose DNA was treated with nuclease

[0084] (- cytotoxin + nuclease) digested DNA from cells treated with cytotoxin (+ cytotoxin + nuclease). Coverage is higher in cells treated with cytotoxin, represented here by the upward deviation from the baseline.

[0085] (C) Simplified representation of the coverage to each other

[0086] (blue = randomly fragmented DNA, orange = + cell toxin + Mnase digestion, gray = - cell toxin + Mnase digestion)

[0087] Figure 6 from (8) shows the relationship between the cfDNA coverage, the preferred end coordinates, the window protection score and the open chromatin regions.

[0088] When pools of fragmented DNA (here plasma DNA molecules) are aligned (also called sequence alignment; sequence alignment refers to the methodical comparison of two or more nucleotide or amino acid sequences in a linear sequence), their ends cluster at specific genomic locations, the so-called preferred end sites, which can vary between DNA molecules originating from different tissues. The "window protection score" (calculated as the number of complete fragments minus the number of fragment endpoints within a given "window") provides information about DNA protection against nuclease digestion, from which nucleosome positioning can be inferred. Genomic coverage reflects the chromatin structure of the tissue of origin.

[0089] Literature:

[0090] 1. Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J. : Cell- free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin . Cell 2016;164:57-68

[0091] 2. Jiang P, Sun K, Tong YK, Cheng SH, Cheng THT, Heung MMS, et al. : Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma. Proc Natl Acad Sei U S A

[0092] 2018 ; 115 : E10925-33.

[0093] 3. Ivanov M, Baranova A, Butler T, Spellman P, Mileyko V. : . Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation. BMC Genomics 2O15;16:S1

[0094] 4. Hardham, A.R., Gunning, B.E.S. Some effects of colchicine on microtubules and cell division in roots of Azolla pinnata . Protoplasma 102, 31-51 (1980) .

[0095] 5. Dane, F., Dalgig, Ö. The Effects of Fungicide Benomyl (Benlate) on Growth and Mitosis in Onion (Allium cepa L.) Root

[0096] Apical Meristem. BIOLOGIA FUTURA 56, 119-128 (2005) 6. Sun K, Jiang P, Cheng SH, Cheng THT, Wong J, Wong VWS, Ng SSM, Ma BBY, Leung TY, Chan SL, Mok TSK, Lai PBS, Chan HLY, Sun H, Chan KCA, Chiu RWK, Lo YMD. Orientat ion-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin. Genome Res. 2019 Mar;29(3) :418-427. doi: 10.1101 / gr.242719.118. PMID: 30808726; PMCID: PMC6396422.

[0097] 7. Sims, David; Sudbery, Ian; Ilott, Nicholas E.; Heger, Andreas; Ponting, Chris P. (2014) . "Sequencing depth and Coverage: key considerations in genomic analyses". Nature Reviews Genetics. 15 (2) : 121-132. Doi : 10.1038 / nrg3642. PMID 24434847. S2CID 13325739.

[0098] 8. Y. M. Dennis Lo, Diana S. C. Han, Peiyong Jiang and Rossa W. K. Chiu (2021) . "Epigenetics, f ragmentomics , and topology of cell-free DNA in liquid biopsies" Science; Vol 372, Issue 6538; DOI: 10.1126 / science . aaw3616

Claims

Patent claims 1. Method for validating a sequencing method for the determination of cfDNA, characterized in that i.) a reference material is provided which is produced by the following steps: a.) providing a cell culture comprising cells, b.) treating the cell culture from a.) with a cell division inhibitor, c.) isolating the nucleic acids comprising nucleosomes, d.) treating the nucleic acids comprising nucleosomes with nucleases or ultrasound, e.) isolating nucleic acids with a length of 20-750 bp, and ii.) the nucleic acids from e.) are determined by means of a sequencing method.

2. A method for validating a sequencing method for the determination of cfDNA according to claim 1, wherein the cell culture comprising cells is eukaryotic cells, mammalian cells or human cells.

3. A method for validating a sequencing method for the determination of cfDNA according to any one of the preceding claims, wherein the cell culture comprising eukaryotic cells is one or more specific cell types selected from the group of body cells, stem cells, B cells, T cells, natural killer cells, diseased or healthy cells, tumor cells, polyclonal cells, monoclonal cells, immortalized cells.

4. A method for validating a sequencing method for the determination of cfDNA according to one of the preceding claims, wherein the cell division inhibitor is selected from the group of mitosis inhibitors, checkpoint inhibitors, in particular selected from the group of colcemide (CAS number: 477-30-5), benomyl (GAS number: 17804-35-2), nocodazole (GAS number: 31430-18-9), paclitaxel (CAS number: 33069-62-4), vincristine (CAS number: 57-22-7), vinblastine (CAS number: 865-21-4), 8-chloroadenosine (CAS number: 34408-14-5), WYE 687 dihydrochloride (CAS number: 1702364-87-1) , AZD 5438 (CAS number: 602306-29-6) , Pladienolide B (CAS number: 445493-23-2) , Temsirolimus (CAS number: 162635-04-3) , Tosyl-L-Arginine Methyl Ester (TAME) (CAS number: 1784-03-8) , YC 1 (CAS number: 170632-47-0) , Indisulam (CAS number: 165668-41-7) .

5. Method for validating a sequencing method for the determination of cfDNA according to one of the preceding claims, wherein the nuclease is selected from the group MNase (EC number: 3.1.31.1), DNaselL3 (EC number: 3.1.21.1), DNase I (EC number: 3.1.21.1).

6. A method for validating a sequencing method for the determination of cfDNA according to one of the preceding claims, characterized in that a validation calibration curve is obtained.

1. A method for validating a sequencing method for determining cfDNA according to one of the preceding claims, characterized in that a sequencing or method selected from the group consisting of pyrosequencing, sequencing by hybridization, ion semiconductor DNA sequencing system, nanopore sequencing, next generation sequencing (NGS), sequencing-by-synthesis (SBS), polymerase chain reaction (PCR), qPCR (real-time quantitative polymerase chain reaction), digital droplet PCR (ddPCR) is carried out. 8 . A method for validating a sequencing method for the determination of cfDNA according to one of the preceding claims, characterized in that the isolated nuncleic acids according to claim 1 e . ) form a validation sample.

9. A method for diagnosing a disease, wherein cfDNA is determined, in particular for assessing the degree of severity and for assessing the course of the disease and making a therapy decision, characterized in that calibration or validation is carried out by means of a method according to one of claims 1 to 8.

10. A method for detecting minimal residual disease, wherein cfDNA is determined, characterized in that calibration or validation is carried out by means of a method according to one of claims 1 to 9.

11. Use of a kit for carrying out a method according to one of the preceding claims, in particular for carrying out a liquid biopsy.

12. Use of a kit for carrying out a method according to claim 11 comprising a validation sample according to claim 8.

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