Preparation of cell-free fragmented nucleic acids for genetic analysis sequencing

Abstract

The present invention relates to a method for the preparation of cell-free fragmented nucleic acids for genetic analysis sequencing, providing a biological sample comprising the cell-free fragmented nucleic acids to be prepared, suitably repairing the damages of the cell-free fragmented nucleic acids that were introduced by a prior fragmentation, fixation, storage, and / or extraction process, and performing a suitable magnetic bead clean-up of the repaired nucleic acids, in order to purify the repaired cell-free fragmented nucleic acids. Based on the preparation of the cell-free fragmented nucleic acids, the invention allows for the improved preparation of a library and an improved sequencing depth of the cell-free fragmentated nucleic acids, including concomitant whole-genome and high-depth targeted sequencing, with low- input concentrations in a biological sample as less than about 0.4 ng / µl. The present invention also provides a kit for the above-mentioned methods.

Description

[0001] Preparation of cell-free fragmented nucleic acids for genetic analysis sequencing

[0002] The present invention relates to a method for the preparation of cell-free fragmented nucleic acids for genetic analysis sequencing, providing a biological sample comprising the cell-free fragmented nucleic acids to be prepared, suitably repairing the damages of the cell-free fragmented nucleic acids that were introduced by a prior fragmentation, fixation, storage, and / or extraction process, and performing a suitable magnetic bead clean-up of the repaired nucleic acids, in order to purify the repaired cell-free fragmented nucleic acids. Based on the preparation of the cell-free fragmented nucleic acids, the invention allows for the improved preparation of a library and an improved sequencing depth of the cell-free fragmentated nucleic acids with low-input concentrations in a biological sample as less than about 0.4 ng / pl. The present invention also provides a kit for the above-mentioned methods.

[0003] Background of the invention

[0004] Cell-free fragmented nucleic acids, such as cell-free DNA (cfDNA) or RNA refer to nucleic acid fragments circulating within the peripheral blood (14). These fragments are small with an average size of around 166 base pairs depending on their origin (15). It is assumed that the fragments of cell-free nucleic acids are primarily released into the bloodstream as a result of cell death (16) providing the information about cell death in the body through quantification and genetic analysis of cell-free nucleic acids.

[0005] For example, the concentration of cfDNA in the blood plasma of healthy individuals is typically between one and 10 ng / ml (17). The concentration of the cfDNA can be significantly higher in diseased individuals. For example, in colorectal cancer (CRC) patients, the concentration can range from 10 to 100 ng / ml (17). The same is true for the size of cfDNA segments, which vary between healthy and diseased individual, with CRC patients having significantly smaller fragments of cfDNA in their blood compared to healthy individuals.

[0006] During pregnancy, between 10 and 20 percent of the cfDNA in maternal peripheral blood is derived from the apoptotic trophoblast cells of the placenta (18). This cfDNA is detectable from around seven weeks of gestation (18). cfDNA can also be derived from microorganisms causing infections in humans, such as viruses (19).

[0007] Genomic mutations and epigenomic aberrations detected in tumors enable a diagnosis, prognosis and, in some cases, permit selection of the most suitable treatment for each patient. Therefore, tumor profiling through DNA sequencing is the cornerstone of cancer precision medicine at present. However, tumor biopsy is not always possible (e.g., in the case of brain tumors in children), and a single tumor biopsy is limited to capture the genetic heterogeneity of a tumor mass, or the set of tumors present in the metastatic setting.

[0008] As opposed to tissue-based molecular profiling, minimally invasive molecular profiling using liquid biopsy analysis can provide a powerful alternative to circumvent tumor sampling challenges, thereby allowing earlier detection of disease relapse and genomic tumor profiling for treatment stratification and enrolment in clinical trials.

[0009] Liquid biopsies refer to the collection of body fluids, primarily blood, but also cerebrospinal fluid (CSF) or urine, for the detection and molecular analysis of cfDNA, which refers as stated above to the fragmented genomic DNA that is secreted into biological fluids from dying tissues and cells. In cancer patients, cfDNA also contains small proportions of circulating tumor- derived DNA (ctDNA). Molecular profiling of ctDNA using DNA sequencing methods has already shown promising potential for precision medicine in common adult malignancies (1, 2), as well as in some pediatric cancers (3).

[0010] For example, analysis of ctDNA can be used for the detection of cancer-specific single nucleotide variants (SNVs) and somatic copy number aberrations (SCNAs) and may reveal more informative insights into the genomic complexity and heterogeneity of tumors in comparison to traditional small tissue needle-biopsies (4, 5). In addition, liquid biopsy analysis of blood is highly amenable to serial sampling, thus facilitating longitudinal monitoring of treatment response and disease evolution in a minimally invasive manner.

[0011] Initial liquid biopsy methods focused on the detection of a small set of selected somatic mutations (generally small mutations, such as SNVs or indels) through targeted sequencing. However, these methods show low sensitivity due to very low concentrations of ctDNA in the blood. Specifically, methods focused on detecting selected mutations (e.g., mutations recurrently detected in tumors or mutations identified by sequencing a tumor from the same patient undergoing liquid biopsy analysis) are limited by the sensitivity of sequencing technologies. For a proper detection of these selected mutations a sampling of DNA molecules is required which include these mutations.

[0012] However, it might be challenging or even impossible to sample the DNA molecules containing the mutations when the concentration of ctDNA in cfDNA is low, or the genomic regions harbouring the mutations are prone to nuclease activity, and thus, not present in cfDNA. In addition, methods focused on detecting a subset of small mutations show low specificity, as oncogenic mutations can also be found in benign clonal expansions of indeterminate tumorigenic potential (6).

[0013] In addition, current methodologies for liquid biopsy analysis rely on conventional short-read sequencing technologies based on sequencing-by-synthesis, of which the most dominant platform is Illumina. During library preparation, the DNA is sheared into small fragments that are amplified and then sequenced, resulting in millions of short reads which are typically 50- 150 bp in length. However, short-read sequencing methods do not deliver long sequencing reads, which are highly informative for the detection of cancer and other diseases (7), and require DNA amplification, thus erasing DNA modifications, such as DNA methylation.

[0014] In fact, as opposed to approaches based on the detection cancer-specific genomic mutations, other liquid biopsy analysis methods rely on the detection of methylation aberrations. These methods are based on bisulphite sequencing of cfDNA or reduced representation bisulfite sequencing (RRBS). For example, multi-cancer early detection assays, such as the Galleri assay from GRAIL, can detect multiple cancer types using a pan-cancer machine learning classifier trained on methylation profiles detected in cfDNA using targeted methylation (8). While methylation-based cfDNA analysis might be useful to predict the anatomical location of tumors, accurate disease classification and treatment selection require the detection of pathognomonic tumor mutations (e.g., canonical gene fusions). Therefore, there is a need for multi-modal methods capable of detecting the multiple types of mutations occurring in tumors and other diseases for accurate diagnosis and disease monitoring.

[0015] Finally, from a deployment standpoint, current liquid biopsy methods are limited due to (a) high upfront costs associated with current sequencing technologies; and (b) low turnaround times due to the need to perform sequencing in a centralized fashion. These financial and technical challenges drastically limit large-scale implementation of ctDNA methods in most health-care settings.

[0016] In contrast, emerging long-read sequencing approaches, such as the Oxford Nanopore Technology (ONT) platform, are capable of reading long, individual native DNA molecules in real time without the need for preamplification steps. This allows simultaneous detection of alterations in the DNA sequence, such as copy -number aberrations (CNAs), structural variants (SVs), and SNVs, as well as DNA modifications (i.e., DNA methylation) from the same assay (9-12).

[0017] This multi-modality is highly beneficial for the analysis of ctDNA from liquid biopsies as it significantly improves testing sensitivity and specificity and allows disease stratification from methylation profiles (13). Compared to other sequencing platforms, nanopore sequencers are also more affordable and cost-effective, highly scalable, and orders of magnitude smaller, which makes them highly portable and easy to deploy in low-resource settings at minimal upfront investment.

[0018] In addition, real-time nanopore sequencing facilitates data analyses to be coupled to the sequencing process, which significantly reduces turn-around times from several weeks to hours. This is critical in clinical practice as long turnaround times delay therapeutic intervention, resulting in late enrolment in clinical trials and alternative treatment regimens. Several proof- of-principle studies have now demonstrated the feasibility of nanopore sequencing for the genomic profiling of tumours (11, 12).

[0019] WO 2023 / 067597 Al discloses methods for determining a tissue of origin, cell type of origin, origination from a cancerous cell or a combination thereof of cell free DNA. The methods comprise the providing of cfDNA, a passing through a nanopore sequencer to produce a sequence with methylation and / or hydroxymethylation data and identifying for the cfDNA the said origins.

[0020] WO 2023 / 235379 Al discloses methods for detecting a molecule of tumor DNA (tDNA) in a sample of cell-free DNA (cfDNA). The method comprises sequencing the sample of cfDNA using a single molecule sequencing to obtain sequence reads. The sequence reads are further analyzed by for differentially methylated CpG sites, the differentially methylated CpG sites having different methylation status in a cancer cell versus a non-cancer cell.

[0021] WO 2021 / 110987 Al discloses methods and apparatuses for estimating the probability of a subject to be affected with cancer, diagnosing cancer, determining the origin of a tumor in a subject and determining a personalized course of treatment in a subject affected or likely to be affected with cancer, based on the sequencing of cell-free nucleic acids and identification therein of genetic, epigenetic, transcriptomic, metabolic and metagenomic biomarkers.

[0022] US 2017 / 0044606 Al discloses methods of determining a nucleic acid sequence. The methods comprising receiving a plurality of DNA fragments, concatemerizing a first set of the DNA fragments to obtain a concatemer and performing single molecule sequencing of the concatemer to obtain a first sequence of the concatemer.

[0023] WO 2021 / 161192 Al discloses a bi sulfite-free, long-read, base-resolution method named long- read TAPS (IrTAPS) for detecting 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in a nucleic acid sequence. IrTAPS comprises mild enzymatic and chemical reactions to detect 5mC and 5hmC, the two major epigenetic marks found in the mammalian genome, quantitatively at base-resolution without affecting unmodified cytosine.

[0024] US 2020 / 0109456 Al discloses a method for detecting circulating tumor DNA (ctDNA) in a sample comprising obtaining a methylation sequence for a sample, identifying at least one CpG Island on the methylation sequence, calculating the proportion of concordantly methylated reads (PMR) for the identical CpG Island, and comparing said PMR to a control background of a normal tissue, wherein the presence of ctDNA is detected in the sample when the PMR of the sample is larger than the control background.

[0025] However, the potential for biological samples, for example liquid biopsy analysis using nanopore sequencing, in particular, remains largely untapped. Primarily due to the lack of experimental preparation methods for cell-free fragmented nucleic acids, such as cfDNA compatible with low concentrations of the cell-free fragmented nucleic acids in a biological sample such as liquid biopsies provided by a subject.

[0026] The invention presented here addresses this challenge by providing an experimental preparation method for cell-free fragmented nucleic acids that enables cost-effective multi-modal analysis of low concentrations of nucleic acids using multiplexed nanopore sequencing. Other objects and advantages will readily become apparent for the person of skill from studying the following more detailed description and examples.

[0027] In a first aspect of the present invention, the problem of the present invention is solved by providing a method for the preparation of cell-free fragmented nucleic acids for genetic analysis sequencing, the method comprising the steps of providing a biological sample comprising the cell-free fragmented nucleic acids to be prepared, suitably repairing the damages of the cell- free fragmented nucleic acids that were introduced by a prior fragmentation, fixation, storage, and / or extraction process, and performing a suitable magnetic bead clean-up of the repaired nucleic acids, in order to purify the repaired cell-free fragmented nucleic acids.

[0028] Preferred is the method according to the present invention, wherein the cell-free fragmented nucleic acids are DNA and / or RNA.

[0029] In the context of the present invention, the term “cell-free” relates to nucleic acids which are fragmented and released into body fluids as a result of cell death providing the information about cell death in the body through quantification and genetic analysis of these cell-free nucleic acids.

[0030] In the context of the present invention the body fluids are used as biological samples from a subject.

[0031] Further preferred is the method according to the present invention, wherein the biological sample is a liquid biopsy selected from the group consisting of blood, plasma, serum, urine, ascites fluid, cerebrospinal fluid (CSF), synovial fluid, and pleural fluid.

[0032] Further preferred is the method according to the present invention, wherein the cell-free fragmented nucleic acids are low-input nucleic acids, for example having a concentration of less than about 10 ng / pl, preferably less than about 5 ng / pl, more preferably less than about 1 ng / pl, even more preferably of less than about 0.4 ng / pl.

[0033] In view of the results as obtained in the experiments as performed in the context of the present invention, the inventors developed a preparation method for cell-free fragmented nucleic acids with concentrations less than about 0.4 ng / .1 for genetic analysis sequencing for example through a nanopore.

[0034] In the context of the present invention, the term ’’about” shall include a deviation of + / - 10% from the value as given.

[0035] In the context of the present invention, the term “less than about 10 ng / pl” shall define a range between 0.1 ng / pl to 10 ng / pl, the term “less than about 5 ng / pl” shall define a range between 0.1 ng / pl to 5 ng / pl, the term “less than about 1 ng / pl” shall define a range between 0.1 ng / pl to 1 ng / pl, the term “less than about 0.4 ng / pl” shall define a range between 0.1 ng / pl to 0.4 ng / pl.

[0036] In the context of the present invention, providing a biological sample comprises providing a biological sample from a subject and extracting cell-free fragmented nucleic acids from said sample.

[0037] In the context of the present invention, a “subject” relates to a mammal, preferably a human, such as a person suffering from a disease such as for example cancer, a person suffering from an infection with a microorganism or a pregnant woman.

[0038] Further preferred is the method according to the present invention, wherein said repairing comprises converting the cell-free fragmented nucleic acids into repaired nucleic acids having 5' phosphorylated, 3' dA-tailed ends, for example with an end-preparation (end-prep) nucleic acid step, and / or repairing the cell-free fragmented nucleic acids, for example with an formalin- fixed, paraffin-embedded (FFPE) nucleic acid repair step and an incubation of the steps at 20 °C for 12 minutes and 65 °C for 12 minutes.

[0039] In a preferred embodiment of the method according to the present invention said repairing of damages of the cell-free fragmented nucleic acids comprises the mixing of extracted cell-free fragmented nucleic acid sample, a FFPE nucleic acid repair buffer, FFPE nucleic acid repair mix, end-prep reaction buffer and end-prep enzyme mix in a volume ratio 1 to 0.07 to 0.04 to 0.07 to 0.06 and incubating at 20 °C for 12 minutes and 65°C for 12 minutes. Further preferred is the method according to the present invention, wherein the magnetic bead clean-up comprises mixing of suitable magnetic beads with the repaired nucleic acid sample in a volume ratio of 1 to 1, incubating the mixture for 10 minutes for a binding of repaired nucleic acids to the magnetic beads, washing the beads twice with 80% ethanol, and eluting the repaired nucleic acids in a suitable buffer.

[0040] The method for the preparation of cell-free fragmented nucleic acids according to the invention comprises an additional suitable magnetic bead clean-up of the repaired nucleic acids compared to standard preparation methods. The performance of a magnetic clean-up step following the repairing of the extracted nucleic acids enables a purification of the repaired cell-free fragmented nucleic acids prior to the production of a library of the cell-free fragmented nucleic acids.

[0041] In the context of the present invention the term “purification” shall mean an accumulation of nucleic acids using paramagnetic beads with functionalized silica surfaces to selectively bind nucleic acids and separate the nucleic acids from the aqueous phase with a magnet. The magnetic beads allow to bind and therefore pull out all the nucleic acids from the aqueous phase and additionally to purify the nucleic acid from the used enzymes in the repairing step through selectively binding of the nucleic acids.

[0042] The purification improves the workflow and the yield efficiency of the nucleic acids and therefore enables to elute the purified nucleic acids in a higher elution volume at the end of the magnetic clean-up. The higher nucleic acid sample volume prior to the preparation of the library enables the performance of more amplification and sequencing analysis steps with the provided biological sample. Furthermore, the preparation according to the invention also allows for an improved sequencing depth of the nucleic acids provided in the biological samples compared to preparation methods known by the person skilled in the art. The sequencing depth of the inventive library preparation is preferably higher than with the standard method (e.g. the ONT Native Barcoding Kit 96 V14, see also below).

[0043] The higher nucleic acid sample volume of purified nucleic acids enables an amplification of one fraction of the nucleic acid sample. Preferred is the method according to the present invention, wherein after the magnetic bead clean-up, a fraction of the eluted nucleic acid sample is selectively amplified, for example using a preselected panel of genes, such as Oncomine™ Pan-Cancer Cell-Free panel (ThermoFisher, https: / / www.thermofisher.com / order / catalog / product / A37664), wherein after amplification the amplified nucleic acids are purified comprising an additional magnetic bead clean-up according to claim 6, and further an additional end-preparation (end-prep) nucleic acid step.

[0044] In a preferred embodiment of the method according to the present invention said amplification following the magnetic bead clean up comprises one fraction of the eluted nucleic acid sample, in particular a volume fraction between 0.3 and 0.5, preferably 0.45 of the eluted nucleic acid sample. This aliquot is target amplified with custom gene panels, such as Oncomine™ PanCancer Cell-Free panel, wherein the gene panel, a PCR master mix and the eluted nucleic acid sample are mixed in a volume ratio 0.15 to 1.15 to 1, wherein after performing a PCR the amplified nucleic acid is magnetic bead clean-upped according to the invention, wherein an additional end-preparation (end-prep) DNA step is performed per 12 pl DNA sample.

[0045] Therefore, the invention includes the preparation of targeted amplified nucleic acids and not amplified nucleic acids, which is in the context of the present invention “native” nucleic acids. The amplified nucleic acids and native nucleic acids allow to perform both shotgun wholegenome sequencing and targeted sequencing of regions of interest (e.g., cancer genes) of the cell-free fragmentated nucleic acids with low-input concentrations in a biological sample as less than about 0.4 ng / pl.

[0046] In a second aspect of the present invention, the problem of the present invention is solved by producing a library of sequences of cell-free fragmented nucleic acids, the method comprising the steps of performing a method for the preparation of cell-free fragmented nucleic acids according to the invention, and ligating native barcodes to the non-amplified and / or amplified nucleic acids, followed by an additional magnetic bead clean-up according to the invention. The library preparation method is similar to the Ligation Sequencing Kit protocol; gDNA is FFPE repaired (note: DNA repair is skipped when using amplicon input) and end-prepped / dA- tailed using the NEBNext End Repair / dA-tailing module. A unique dT-tailed barcode adapter is then ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end which is used as a hook to ligate to the supplied sequencing adapter. See, for example, https: / / store.nanoporetech.com / us / native-barcoding-kit- 24-vl4.html

[0047] In a preferred embodiment of the method according to the present invention said ligating of native barcodes comprises the ligating of native barcodes to unamplified and / or amplified nucleic acids by mixing nucleic acid samples, native barcodes and blunt / TA ligase master mix in a volume ratio 1 to 0.1 to 1.1, incubating for 30 to 40 minutes at room temperature, stopping the reaction with EDTA and pooling the samples together, followed by a magnetic bead cleanup according to the invention.

[0048] Further preferred is the method according to the present invention, wherein the method comprises ligating preselected sequencing adapters to the barcoded nucleic acids, followed by an additional magnetic bead clean-up according to the invention, wherein the magnetic bead clean-up is performed by mixing adapter ligated nucleic acids with a magnetic beads sample in a volume ratio 1 to 0.8.

[0049] In a preferred embodiment of the method according to the present invention said preselected sequencing adapters are ligated to the barcoded nucleic acids by mixing pooled barcoded nucleic acids, native adapter, (x5) ligation reaction buffer and T4 DNA ligase in a volume ratio 1 to 1 / 6 to 1 / 3 to 1 / 6, incubating for 30 to 40 minutes at room temperature, wherein a 1 to 0.8 magnetic bead clean-up is performed by mixing adapter ligated nucleic acid sample with a magnetic beads sample in a volume ratio 1 to 0.8, incubating the mixture for 10 minutes for binding of the nucleic acids to the magnetic beads, washing the beads twice with short fragment buffer and eluting the adapter ligated nucleic acids into an elution buffer.

[0050] Further preferred is the method according to the present invention, wherein the sequencing depth of the library as produced is increased, for example by a factor of at least 1.5, preferably of at least 2.0, or more when compared to a control library prepared without performing a method according to the present invention.

[0051] In the context of the present invention the term “sequencing depth” is used according to the article of Sims et al., 2014 (20). In a third aspect of the present invention, the problem of the present invention is solved by a method for sequencing the library of cell-free fragmented nucleic acids, comprising the method according to the invention, and further comprising the step of sequencing the cell-free fragmented nucleic acids, preferably nanopore sequencing.

[0052] The sequencing of the library according to the invention can be carried out by any sequencing technology for a library known to a person skilled in the art.

[0053] Further preferred is the method according to the present invention, wherein the method further comprises the step of a genetic analysis of the sequences as obtained.

[0054] Further preferred is the method according to the present invention, wherein the genetic analysis comprises a genetic analysis of nucleic acids derived, for example, from apoptotic trophoblast cells of the placenta during pregnancy, from microorganism cells during infection and from disease-associated cells from a subject.

[0055] Further preferred is the method according to the present invention, wherein the genetic analysis comprises a genetic analysis of disease-associated mutations of DNA, such as somatic copy number aberrations (SCNAs), single nucleotide variants (SNVs), indels, aberrant DNA fragmentation profiles, genome-wide methylation, fragmentomic features, nucleosome profiling, epigenomic mutations, such as 5mC and 5hmC, and preferably a genetic analysis of DNA mutations in cancer cells.

[0056] Further preferred is the method according to the present invention, wherein the disease- associated mutations of nucleic acids comprise diseases characterized by increased inflammation and tissue damage such as cancer, autoimmune conditions, sepsis, Alzheimer's.

[0057] Further preferred is the method according to the present invention, wherein the cell-free fragmented nucleic acids are derived from cancer cells selected from colorectal cancer cells, gastric cancer cells, rectal cancer cells, breast cancer cells, cervical cancer cells, endocervical cancer cells, colon cancer cells, esophageal cancer cells, brain cancer cells, head and neck cancer cells, renal cancer cells, meningeal cancer cells, glioma, glioblastoma, lung cancer cells, mesothelioma, ovarian cancer cells, pancreatic cancer cells, neuroendocrine cancer cells, prostatic cancer cells, skin cancer cells, stomach cancer cells, thyroid cancer cells, uterine cancer cells, and testicular cancer cells.

[0058] Further preferred is the method according to the present invention, wherein the nanopore sequencing is performed with a nanopore sequencer capable of single base pair sequencing resolution and distinguishing between methylated DNA bases, hydroxymethylated DNA bases and unmethylated / unhydroxymethylated DNA bases.

[0059] In some embodiments, the method comprises identifying a cancer-specific DNA modification in a cancer cell. In some embodiments, DNA modification is DNA methylation and / or DNA hydroxymethylation. In some embodiments, DNA methylation is 5 '-methylcytosine modification and / or the DNA hydroxymethylation is a 5 '-hydroxymethylcytosine modification.

[0060] In a fourth aspect of the present invention, the problem of the present invention is solved by providing a kit comprising materials for performing a method according to the invention, such as suitable buffers, master mixes, magnetic beads, native barcodes, native adaptors, and / or cell- free fragmented nucleic acids preparation buffers.

[0061] In a fifth aspect of the present invention, the problem of the present invention is solved by providing the use of the kit for preparing cell-free fragmented nucleic acids, producing a library of sequences of cell-free fragmented nucleic acids, sequencing the library of cell-free fragmented nucleic acids and / or performing a genetic analysis of the sequences as obtained, according to a method according to the invention.

[0062] In a sixth aspect of the present invention, the problem of the present invention is solved by providing a method for treating a subject that provided a biological sample, comprising the method according to the invention, the method for sequencing cell-free fragmented nucleic acids according to the invention, and providing a suitable therapy to the subject based on the genetic analysis, preferably providing a therapy selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, hormone therapy, and targeted therapy.

[0063] In a seventh aspect of the present invention, the problem of the present invention is solved by providing a method for providing a prognosis for the risk for a disease based on genetic analysis of the cell-free fragmented nucleic acids, comprising the method according to the invention, the method for sequencing according to the invention, and further comprising the step of providing a prognosis for the subject based on the genetic analysis in a subject as determined.

[0064] In an eighth aspect of the present invention, the problem of the present invention is solved by providing a method for monitoring the status of a disease based on genetic analysis in a subject, comprising the method according to the present invention, the method for sequencing according to the present invention, and further comprising the step of monitoring the status of a disease based on genetic analysis in a subject based on comparing the differences in the genetic analysis in the obtained biological samples as determined.

[0065] In a nineth aspect of the present invention, the problem of the present invention is solved by providing a method for identifying a subject having a disease based on genetic analysis in need of therapy, comprising the cell-free fragmented nucleic acids preparation according to the present invention, the genetic analysis sequencing according to the present invention, and further comprising the step of identifying a subject having a disease based on genetic analysis in need of therapy.

[0066] A major advantage of the invention is that the invention allows to perform both (a) shotgun whole-genome sequencing of native cell-free fragmented nucleic acids, and (b) targeted sequencing of selected genes on the same biological sample input material. This facilitates the simultaneous detection and analysis of copy number aberrations (CNAs), genome-wide methylation, fragmentomic features and nucleosome profiling, as well as cancer-specific single nucleotide variants (SNVs), which can have important clinical implications for accurate diagnosis, and treatment stratification, for a low concentration of cell-free fragmented nucleic acids in samples.

[0067] In addition, the invention permits multiplexing of samples in a single nanopore sequencing flowcell. This allows to reduce the per-sample cost to ~£30, which is at least one order of magnitude lower than existing technologies for cell-free fragmented nucleic acids, such as cfDNA profiling.

[0068] The invention has applications for the analysis of disease-associated mutations of cell-free fragmented nucleic acids sequence (including mutations, copy number aberrations, structural variants, indels, aberrant DNA fragmentation profiles) and epigenome (including DNA modifications, such as 5mC and 5hmC) for the detection or monitoring of diseases, such as cancer.

[0069] The invention permits to obtain at >2x sequencing yield per flowcell and sample as compared to existing preparation methods of DNA for genetic analysis sequencing, such as the standard preparation method for cfDNA developed by Oxford Nanopore Sequencing technologies (Oxford, UK) for nanopore sequencing.

[0070] The present invention relates to the following items:

[0071] Item 1. A method for the preparation of cell-free fragmented nucleic acids for genetic analysis sequencing, the method comprising the steps of

[0072] (a) providing a biological sample comprising the cell-free fragmented nucleic acids to be prepared,

[0073] (b) suitably repairing the damages of the cell-free fragmented nucleic acids that were introduced by a prior fragmentation, fixation, storage, and / or extraction process, and

[0074] (c) performing a suitable magnetic bead clean-up of the repaired nucleic acids, in order to purify the repaired cell-free fragmented nucleic acids.

[0075] Item 2. The method according to item 1, wherein the cell-free fragmented nucleic acids are DNA and / or RNA.

[0076] Item 3. The method according to item 1 or 2, wherein the cell-free fragmented nucleic acids are low-input nucleic acids, for example having a concentration of less than about 10 ng / pl, preferably less than about 5 ng / pl, more preferably less than about 1 ng / pl, even more preferably of less than about 0.4 ng / pl.

[0077] Item 4. The method according to any one of items 1 to 3, wherein the biological sample is a liquid biopsy selected from the group consisting of blood, plasma, serum, urine, ascites fluid, cerebrospinal fluid (CSF), synovial fluid, and pleural fluid.

[0078] Item 5. The method according to any one of items 1 to 4, wherein said repairing comprises converting the cell-free fragmented nucleic acids into repaired nucleic acids having 5' phosphorylated, 3' dA-tailed ends, for example with an end-preparation (end-prep) nucleic acid step, and / or repairing the cell-free fragmented nucleic acids, for example with an formalin- fixed, paraffin-embedded (FFPE) nucleic acid repair step and an incubation of the steps at 20 °C for 12 minutes and 65°C for 12 minutes.

[0079] Item 6. The method according to any one of items 1 to 5, wherein the magnetic bead clean-up comprises mixing of suitable magnetic beads with the repaired nucleic acid sample in a volume ratio of 1 to 1, incubating the mixture for 10 minutes for a binding of repaired nucleic acids to the magnetic beads, washing the beads twice with 80% ethanol, and eluting the repaired nucleic acid in a suitable buffer.

[0080] Item 7. The method according to any one of items 1 to 6, wherein after the magnetic bead cleanup, a fraction of the eluted nucleic acid sample is selectively amplified, for example using a preselected panel of genes, such as Oncomine™ Pan-Cancer Cell-Free panel, wherein after amplification the amplified nucleic acids are purified comprising an additional magnetic bead clean-up according to item 6, and further an additional end-preparation (end-prep) nucleic acid step.

[0081] Item 8. A method for producing a library of sequences of cell-free fragmented nucleic acids, the method comprising the steps of

[0082] (a) performing a method according to any one of items 1 to 7, and

[0083] (b) ligating native barcodes to the non-amplified and / or amplified nucleic acids, followed by an additional magnetic bead clean-up according to item 6.

[0084] Item 9. The method according to item 8, furthermore comprising ligating preselected sequencing adapters to the barcoded nucleic acids, followed by an additional magnetic bead clean-up according to item 6, wherein the magnetic bead clean-up is performed by mixing adapter ligated nucleic acids with a magnetic beads sample in a volume ratio 1 to 0.8.

[0085] Item 10. The method according to item 8 or 9, wherein the sequencing depth of the library as produced is increased, for example by a factor of at least 1.5, preferably of at least 2.0, or more when compared to a control library prepared without performing a method according to any of items 1 to 7. Item 11. A method for sequencing the library of cell-free fragmented nucleic acids, comprising the method according to any one of items 8 to 10, and further comprising the step of sequencing the cell-free fragmented nucleic acids, preferably nanopore sequencing.

[0086] Item 12. The method according to item 11, further comprising the step of a genetic analysis of the sequences as obtained.

[0087] Item 13. The method according to item 12, wherein the genetic analysis comprises a genetic analysis of nucleic acids derived, for example, from apoptotic trophoblast cells of the placenta during pregnancy, from microorganism cells during infection and from disease-associated cells from a subject.

[0088] Item 14. A kit comprising materials for performing a method according to any one of items 1 to 13, such as suitable buffers, master mixes, magnetic beads, native barcodes, native adaptors, and / or cell-free fragmented nucleic acids preparation buffers.

[0089] Item 15. Use of the kit according to item 14 for preparing cell-free fragmented nucleic acids, producing a library of sequences of cell-free fragmented nucleic acids, sequencing the library of cell-free fragmented nucleic acids and / or performing a genetic analysis of the sequences as obtained, according to a method according to any one of items 1 to 13.

[0090] The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.

[0091] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the terminology employed herein is for the purpose of description and not for limitation. Therefore, while the embodiments herein have been described as preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein. For the purposes of the present invention, all references as cited are herewith incorporated by reference in their entireties.

[0092] Figure 1 shows the comparison between the existing method for the preparation of cfDNA for nanopore sequencing (left) and the one presented in this document (right; changes are highlighted in bold and underlined).

[0093] Figure 2 shows the target amplification step for preparation of the cfDNA for targeted sequencing of selected genes with a nanopore sequencer. As indicated in Figure 1, this follows the DNA Repair and End-Prep step, where a fraction of 0.56 of the Repair and End-Prep sample are taken forward for barcoding and low-coverage whole genome sequencing, and the remaining fraction of 0.44 are used for the amplification step and targeted sequencing as outlined in this figure.

[0094] Figure 3 shows the validation of a multiplexing procedure using samples spanning a DNA input ranging from 5 to 150ng in 12 pl biological sample. (A) Schematic illustrating the experimental steps used to generate barcoded DNA samples from DNA extracted from cell line NA12878. (B) Sequencing yield quantified as coverage of the human genome computed using Mosdepth (y axis) achieved for each input sample. Each input sample was run in triplicates (three experimental repeats) and compared to two different ONT protocols. “Original protocol” refers to the ONT Native Barcoding Kit 96 V14. “ONT cfDNA protocol” refers to the ONT human blood cfDNA v5 (November 2023) protocol. (C) Correlation between the input DNA (x axis) and the sequencing yield quantified as coverage of the human genome computed using Mosdepth (y axis). Overall, the sequencing output per sample is proportional to the input DNA across all three methods / protocols. (D) Direct comparison of sequencing yield for each input sample of the invention method with the two ONT protocols / methods. Combined, sequencing yield and performance is significantly higher in the invention method compared to the two ONT protocols.

[0095] Figures 4 and 5 shows that targeted amplification and nanopore sequencing of cfDNA permits detection of mutations at low variant allele fraction in target genomic regions which are sequenced to high depth. Figure 5 (A) Sequencing depth for each target included in the Oncomine™ Pan-Cancer Cell-Free panel by target gene across three truth-set control samples (SeraSeq ctDNA 5%, 1% and 0.1% allele fraction). (B) Total sequencing depth achieved following target amplification and sequencing for the above three SeraSeq truth-set control samples. (C) Variant allele fractions detected for genes with known mutations from the SeraSeq truth-set control samples. (D) Mean variant allele fraction across all known mutations (point) in comparison to true variant allele fraction (triangle) for each of the three SeraSeq truth-set control samples.

[0096] Examples

[0097] Example 1: Preparation method for low concentrated cell-free fragmented nucleic acids

[0098] This method consists of two alternatives after the extraction of fragmented DNA, such as, for example, cell free DNA (cfDNA) (Part A). The first alternative includes barcoding and nanopore whole-genome sequencing of native cfDNA (Part B). The second alternative includes amplification and subsequent target amplicon sequencing of cfDNA using a user-defined panel (Part C). To show proof of principle, the inventors herein used the Oncomine™ Pan-Cancer Cell-Free panel. However, the inventive method is compatible with any other gene panel. Finally, resulting cfDNA libraries from Part B and C are nanopore sequenced on PromethlON flowcells (Part D).

[0099] Part A. DNA extraction

[0100] The first step consists of extracting nucleic acids, such as cfDNA from the sample using a commercial kit, such as the QIAamp DSP Circulating NA kit (Cat. No. / ID: 61504; Qiagen), eluting the extracted cfDNA into Eppendorf DNA LoBind® Tubes (Eppendorf).

[0101] In some embodiments, the method comprises the elution of extracted DNA into 12 pl elution buffer.

[0102] Part B. Whole-genome sequencing and barcoding of native cfDNA

[0103] The inventive method for barcoding the native cfDNA and whole genome nanopore sequencing is described below and illustrated in Figures 1-2. A detailed comparison of the here described method with the current method designed by Oxford Nanopore Technologies is shown in Figure 1. B.l DNA Repair and End Prep

[0104] An Ultra II End Repair / dA-Tailing preparation is used to convert the fragmented DNA into repaired DNA having 5' phosphorylated, 3' dA-tailed ends.

[0105] Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples is a common practice. However, the methods used for this fixation and storage significantly damage and compromise the quality of DNA from these samples. The same is true for an extraction of DNA, which also damages the DNA. As a result, it is necessary to include a FFPE DNA repair step of DNA to obtain useful information, including high-quality sequence data, especially when sample amounts are limited. An example for such an FFPE repair kit suitable for the method according to the present invention is NEBNext FFPE Kit (NEB, www.neb.com).

[0106] In some embodiments, following cfDNA extraction and quality control, the DNA end repair mastermix is prepared by mixing 0.875pl of NEBNext FFPE DNA Repair buffer, 0.875pl Ultra II End-Prep Reaction buffer, 0.75pl Ultra II End-Prep enzyme mix and 0.5 pl NEBNext FFPE DNA repair mix, which are added to 12 l of extracted sample cfDNA, respectively.

[0107] Following gentle pipette mixing and spinning down, the reaction is then incubated at 20°C for ~12 mins, followed by a further incubation at 65°C for ~12mins.

[0108] Next, a 1 : 1 AMPure XP magnetic bead clean-up is performed by mixing of AMPure XP beads sample with the repaired cfDNA sample in a ratio 1 :1, followed by incubating the mixture for 10 minutes for a binding to the magnetic beads, magnetic separation of the beads, washing the beads twice with 80% ethanol and eluting the fixed end-repaired cfDNA into 40 pl molecular grade nuclease-free water, in order to ensure a sufficient sample volume for simultaneous shotgun WGS and additional targeted sequencing (Part C of the method described below). However, if required for very low input samples (e.g., < lOng input DNA at the start), the elution volume can be lowered to 25 pl.

[0109] In some embodiments the magnetic bead clean-up results in a 40 pl, no less than 25 pl, repaired DNA sample volume. When compared to a standard protocol without the magnetic bead clean up where only 15 pl fixed end-repaired DNA final volume are obtained, whereas in the present method 25 pl, preferably 40 pl, of repaired DNA final sample volume enables sample volumes suitable for shotgun whole-genome sequencing (WGS), and an additional target amplification for targeted sequencing of selected genes. Preferably, the sample volume for the WGS is 22.5 pl and the sample volume for targeted amplification is 17.5 pl.

[0110] As mentioned above, the method developed by ONT does not include a bead clean up step. As a result, the final volume of sample obtained with the method developed by ONT is 15 pl, disadvantageously resulting in less than Ipl to be used in Step B.2.

[0111] B.2 Native Barcode Ligation

[0112] The native barcoding is a method providing unique barcodes, for example 96, to enable PCR- free multiplexing of dsDNA samples such as gDNA and amplicons.

[0113] For the library preparation, the repaired and dA-tailed DNA is ligated to a unique dT-tailed barcode adapter. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end, and this is used as a hook to ligate to the supplied sequencing adapter when used for sequencing. This method achieves modal sequencing accuracies of over 99% with high output on a nanopore sequencer.

[0114] Native barcoding is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, for example, using a Short Fragment Eliminator). Deconvolution of barcoded sequencing data includes classifying the barcode sequence and sorting the results into corresponding folders.

[0115] Following DNA end repair, 22.5pl of end-repaired DNA (from step B. l) are mixed with 2.5pl of a unique barcode (NB01-96 from the SQK-NBD114.96 Native Barcoding kit) and 25pl of Blunt / TA Ligase Master Mix and incubated for ~30 mins at room temperature. 5 pl of EDTA (0.5M) is added to terminate the ligation reaction. Note that the remainder of end-repaired DNA (~ 17.5 l) from step B.l is then set aside for targeted amplicon sequencing as described in Part

[0116] C. Subsequently, barcoded samples to be multiplexed are pooled together and another 1 : 1 AMPure XP bead clean up (with two 80% EtOH washes) is performed, eluting the multiplexed samples in 35 pl of molecular grade nuclease-free water.

[0117] B.3. Adapter ligation Following native barcode ligation, sample pooling for multiplexing and bead clean-up (as described in B.2), 35 pl of barcoded and pooled DNA is mixed with 5 pl of ligation adapter, lOpl of NEBNext Quick ligation reaction buffer (5X), and 5 pl of Quick T4 DNA ligase, and incubated for 30-40 mins at room temperature. Subsequently, adapter-ligated DNA is cleaned up with AMPure XP beads (1 :0.8), washed twice with short fragment buffer (SFB), and eluted into 25 pl of elution buffer.

[0118] Part C. Targeted amplification and sequencing

[0119] As outlined in Step B.2 and Figures 1-2, an aliquot of the remaining end-repaired DNA from Step B.l (-12-17.5 pl) may be used for target amplification and sequencing. To show proof of principle, the inventors used the Oncomine™ Pan-Cancer Cell-Free panel. However, this approach is compatible with other custom gene panels. The repaired DNA from Step B. l is amplified following the manufacturer’s recommendations, using 2pl of the Oncomine PanCancer Panel and 15 pl of PCR master mix and the recommended PCR program.

[0120] Following targeted gene amplification, the resulting DNA was cleaned up using 1 : 1 AMPure XP beads (eluted in 20pl molecular grade nuclease-free H2O) and end-prepped with Ultra II end-prep reaction buffer (1.75pl) and Ultra II end-prep enzyme mix (0.75pl) incubated on a thermal cycler at 20°C for -12 mins followed by a further incubation at 65°C for ~12mins. The end-repaired amplicon DNA is then subjected to Steps B.2 - B.3 for barcoding and adaptor ligation.

[0121] Part D. Multiplex nanopore sequencing

[0122] Barcoded samples for shotgun whole-genome sequencing (Part B) are sequenced using a single PromethlON flowcell. Similarly, the samples prepared for targeted amplification (Part C) are also nanopore sequenced using a single PromethlON flowcell. In both settings, libraries are loaded at 20 fmol, and sequencing is performed over 72hrs using the Short Fragment Mode, setting the minimum read length to 20bp. The flowcells are flushed every 24hrs and libraries reloaded to boost pore occupancy and reduce blockages. The run is conducted with high- accuracy base calling (including calling of 5-methylcytosine (5mC) and 5- hydroxymethylcytosine (5hmC) modified bases). Upon base analysis, sequencing reads are demultiplexed computationally for further analysis, such as alignment to the reference genome, methylation analysis, and mutation detection. Example 2: Efficient sample multiplexing

[0123] Several different dilutions of the DNA extraction solutions were prepared from the range of 5 ng to 150 ng per 12 pl DNA sample for proof of concept of the inventive method for low concentrations of sequencing of cell-free DNA (cfDNA). The inventive method permits to perform both shotgun whole-genome sequencing and targeted sequencing of regions of interest (e.g., cancer genes) from as low as 5ng of cfDNA per 12 pl DNA sample.

[0124] Additionally, the inventive method has the ability to perform both (a) shotgun whole-genome sequencing of native cfDNA, and (b) targeted sequencing of selected genes on the same sample input material. Furthermore, the inventive method permits multiplexing of samples in a single nanopore sequencing flowcell.

[0125] The schematic illustration of the experimental steps used to generate barcoded DNA samples from DNA extracted from cell line NA12878 is shown in Figure 3 A. The correlation between the input DNA (x axis) and the sequencing yield quantified as coverage of the human genome computed using Mosdepth (y axis) is shown in Figure 3B-D. Overall, the sequencing output per sample is proportional to the input DNA, and sequencing yields achieved with the invention method is superior to sequencing output achieved with the two comparable ONT protocols (where “Original protocol” refers to the ONT Native Barcoding Kit 96 V14. “ONT cfDNA protocol” refers to the ONT human blood cfDNA v5 (November 2023) protocol).

[0126] Example 3: Improved data yield as compared to the standard method developed by ONT Several experiments were conducted to compare the data yield of the standard method developed by ONT to the inventive method for an inventive library preparation for wholegenome sequencing (WGS) of cfDNA. The sequencing depth of the inventive library preparation is higher than with the standard method (the ONT Native Barcoding Kit 96 V14).

[0127] Example 4: Shotgun nanopore WGS and targeted sequencing using a single cfDNA sample

[0128] The method presented in this document allows for the concomitant detection of multiple types of mutations. Specifically, the shotgun WGS permits detection of copy number aberrations, fragmentomics, nucleosome occupancy profiles and genome-wide methylation patterns. In addition, the targeted sequencing step enables the detection of mutations, including those present at low variant allele fraction as shown in Figure 4 and 5. References Adalsteinsson, V. A. etal. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nat. Commun. 8, (2017). Wan, J. C. M. et al. Liquid biopsies come of age: Towards implementation of circulating tumour DNA. Nat. Rev. Cancer 17, 223-238 (2017). Stankunaite, R. et al. Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours. Eur. J. Cancer 162, 209-220 (2 2022). Alix-Panabieres, C. & Pantel, K. Clinical applications of circulating tumor cells and circulating tumor DNA as liquid biopsy. Cancer Discovery vol. 6 479-491 Preprint at https: / / doi.Org / 10.l 158 / 2159-8290. CD-15-1483 / 43226 / P / CLINICAL-APPLICATIONS- OF-CIRCULATING-TUMOR-CELLS (5 2016). Mattos- Arruda, L. D. & Caldas, C. Cell-free circulating tumour DNA as a liquid biopsy in breast cancer. Molecular Oncology vol. 10 464-474 Preprint at https: / / doi.Org / 10.1016 / J.MOLONC.2015.12.001 (3 2016). Giesselmann, P. et al. Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing. Nat. BiotechnoL 37, 1478-1481 (12 2019). Yu, S. C. Y. et al. Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma. Proc. Natl. Acad. Sci. U. S. A. 118, e2114937118 (2021). Klein, E. A. et al. Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set. Ann. Oncol. 32, 1167-1177 (2021). Bowden, R. et al. Sequencing of human genomes with nanopore technology. Nat. Commun. 10, (2019). Sakamoto, Y., Sereewattanawoot, S. & Suzuki, A. A new era of long-read sequencing for cancer genomics. J. Hum. Genet. 65, 3-10 (1 2020). Euskirchen, P. et al. Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing. Acta NeuropathoL 134, 691-703 (11 2017). Capper, D. etal. DNA methylation-based classification of central nervous system tumours. Nature 55, (2018). Pickles, J. C. et al. DNA methylation-based profiling for paediatric CNS tumour diagnosis and treatment: a population-based study. The Lancet Child & Adolescent Health 4, 121— 130 (2 2020). Moss, J, et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat Commun. 9(1): 5068 (2018). hi J, et al. Size profile of cell-free DNA: A beacon guiding the practice and innovation of clinical testing. Theranostics. 10(11): 4737-4748 (2020). an JCM, etal. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 17(4): 223-238 (2017). ouliere F, et al. Multi -marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer. Mol Oncol. 8(5): 927-941 (2014). Grace MR, et al. Cell-free DNA screening: complexities and challenges of clinical implementation. Obstet Gynecol Surv. 71(8): 477-487 (2016). De Vlaminck I, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc NatlAcadSci. 112(43): 13336-13341 (2015). Sims D, et al. Sequencing depth and coverage: key considerations in genomic analyses. Nature Reviews Genetics 15: 121-132 (2014).

Claims

Claims1. A method for the preparation of cell-free fragmented nucleic acids for genetic analysis sequencing, the method comprising the steps of:(a) providing a biological sample comprising the cell-free fragmented nucleic acids to be prepared,(b) suitably repairing the damages of the cell-free fragmented nucleic acids that were introduced by a fragmentation, fixation, storage, and / or extraction process, and(c) performing a suitable magnetic bead clean-up of the repaired nucleic acids, in order to accumulate the repaired cell-free fragmented nucleic acids.

2. The method according to claim 1, wherein the cell-free fragmented nucleic acids are DNA and / or RNA.

3. The method according to claim 1 or 2, wherein the cell-free fragmented nucleic acids are low-input nucleic acids, for example having a concentration of less than about 10 ng / pl, preferably less than about 5 ng / pl, more preferably less than about 1 ng / pl, even more preferably of less than about 0.4 ng / pl.

4. The method according to any one of claims 1 to 3, wherein the biological sample is a liquid biopsy selected from the group consisting of blood, plasma, serum, urine, ascites fluid, cerebrospinal fluid (CSF), synovial fluid, and pleural fluid.

5. The method according to any one of claims 1 to 4, wherein said repairing comprises converting the cell-free fragmented nucleic acids into repaired nucleic acids having 5' phosphorylated, 3' dA-tailed ends, for example with an end-preparation (end-prep) nucleic acid step, and / or repairing the cell-free fragmented nucleic acids in the biological sample, for example with an FFPE nucleic acid repair buffer, FFPE nucleic acid repair mix, end-prep reaction buffer and end-prep enzyme mix, preferably in a volume ratio of 1 to 0.07 to 0.04 to 0.07 to 0.06, respectively, and optionally incubating at 20 °C for 12 minutes and 65°C for 12 minutes.

6. The method according to any one of claims 1 to 5, wherein the magnetic bead clean up comprises mixing of suitable magnetic beads with the repaired nucleic acid sample in a volume ratio of 1 to 1, incubating the mixture for 10 minutes for a binding to the magnetic beads, washing the beads twice with 80% ethanol, and eluting the repaired nucleic acid in a suitable buffer.

7. The method according to any one of claims 1 to 6, wherein after the magnetic bead clean-up, a fraction of the eluted nucleic acid sample is selectively amplified, for example using a preselected panel of genes, such as Oncomine™ Pan-Cancer Cell-Free panel, wherein after amplification the amplified nucleic acids are purified comprising an additional magnetic bead clean-up according to claim 6, and further an additional end-preparation (end-prep) DNA step is performed.

8. A method for producing a library of sequences of cell-free fragmented nucleic acids, the method comprising the steps of:(a) performing a method according to any one of claims 1 to 7, and(b) ligating native barcodes to the non-amplified and / or amplified nucleic acids, followed by an additional magnetic bead clean-up according to claim 6.

9. The method according to claim 8, furthermore comprising ligating preselected sequencing adapters to the barcoded nucleic acids, followed by an additional magnetic bead clean-up according to claim 6, wherein the magnetic bead clean-up is performed by mixing adapter ligated nucleic acids with a magnetic beads sample in a volume ratio 1 to 0.8.

10. The method according to claim 8 or 9, wherein the sequencing depth of the library as produced is increased, for example by a factor of at least 1.5, preferably of at least 2.0, or more when compared to a control library prepared without performing a method according to any of claims 1 to 7.

11. A method for sequencing the library of cell-free fragmented nucleic acids, comprising the method according to any one of claims 8 to 10, and further comprising the step of sequencing the cell-free fragmented nucleic acids.

12. The method according to claim 11, further comprising the step of a genetic analysis of the sequences as obtained.

13. The method according to claim 12, wherein the genetic analysis comprises a genetic analysis of nucleic acids derived, for example, from apoptotic trophoblast cells of the placenta during pregnancy, from microorganism cells during infection and from disease-associated cells from a subject.

14. A kit comprising materials for performing a method according to any one of claims 1 to 13, such as suitable buffers, master mixes, magnetic beads, native barcodes, native adaptors, and / or cell-free fragmented nucleic acids preparation buffers.

15. Use of the kit according to claim 14 for preparing cell-free fragmented nucleic acids, producing a library of sequences of cell-free fragmented nucleic acids, sequencing the library of cell-free fragmented nucleic acids and / or performing a genetic analysis of the sequences as obtained, according to a method according to any one of claims 1 to 13.

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