Methods and reagents for double-strand break identification

Abstract

Provided herein are methods, kits, and reagents for the analysis of nucleic acids. In particular, provided herein are methods, kits, and reagents that are relevant to the identification of double strand breaks (DSBs) and optionally epigenetic modifications.

Description

[0001] METHODS AND REAGENTS FOR DOUBLE-STRAND BREAK AND EPIGENETIC MODIFICATION IDENTIFICATION

[0002] FIELD OF THE INVENTION

[0003] Provided herein are methods, kits, and reagents for the analysis of nucleic acids. In particular, provided herein are methods, kits, and reagents that are relevant to the identification of double strand breaks (DSBs) and optionally epigenetic modifications.

[0004] BACKGROUND OF THE INVENTION

[0005] There is a need for methods capable of identifying DSBs within nucleic acid samples. One of the reasons for identifying DSBs is because site-specific endonucleases (such as CRISPR-Cas9-based systems) can cause off- target DSBs and these DSBs can be hazardous to the cell or organism. This is particularly relevant where the sitespecific endonuclease is used as part of a gene therapy technique for the treatment of a human because side effects caused by off-target DSBs are undesirable. Thus, there is a need to be able to identify the presence, location, and frequency of DSBs within a sample.

[0006] The challenge of accurately identifying DSBs within a sample is compounded where a DSB is very low frequency with a large sample. The situation can occur when seeking off-target DSBs within human genomic DNA.

[0007] Many current methods for identifying DSBs rely on sequencing technology that generates short reads. This can be disadvantageous when mapping reads to low complexity regions of a genome and can result in unresolvable regions. Short reads are also less preferred for identifying genomic rearrangements.

[0008] Current methods for identifying DSBs do not allow for the direct detection of epigenetic modifications. The association between epigenetic modifications and DNA breaks is of interest because modifications can influence the activity of nucleases (such as Cas9 in gene-editing) and therefore the combined break / modification data would be beneficial to research and therapeutic development activities such as improving the guide design process.

[0009] Thus, there is a need for improved methods of DSB detection.

[0010] SUMMARY OF THE INVENTION

[0011] In an aspect, there is provided a method of preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the method comprising labelling a DSB end with a sequencing adapter suitable for single-molecule sequencing to generate the nucleic acid library.

[0012] In some embodiments, the method comprises exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends in the nucleic acid sample; and exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate the nucleic acid library.

[0013] The nucleic acid library may be suitable for the direct detection of epigenetic modifications.

[0014] In an aspect, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends; exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0015] In an aspect, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ attachment of a first linker to DSB ends in the nucleic acid sample; exposing the nucleic acid sample to conditions suitable for attachment of a sequencing adapter that is suitable for single-molecule sequencing to the first linker in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0016] In some embodiments, the methods comprise alignment to a reference sequence of sequence reads generated by the sequencing; and the identification of the location of DSB s within the nucleic acid sample. The methods may comprise detecting the presence / absence of epigenetic modifications.

[0017] The method steps recited in the appended claims or statements of invention herein may be performed in order but additional intervening steps may also be performed. For instance, selection and / or purification steps may intervene the recited steps. Additionally, or alternatively, there may be intervening steps to alter the conditions to render the sample suitable for subsequent steps, e.g. cell lysis. Steps prior to the recited steps and steps subsequent to the recited steps may also be performed. Some steps may be performed concurrently, as the skilled person would be aware. For instance, fixation and permeabilization may be simultaneous. End-repairing and tailing may be simultaneous. The adding, annealing, and ligating of a nucleic acid adapter to a nucleic acid sample may performed as a single step. The steps that can be combined and the steps that must be separate is evident to the skilled person in light of the present disclosure.

[0018] In an aspect, there is provided a kit for preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the kit comprising: a linker, and optionally a sequencing adapter suitable for singlemolecule sequencing. The kit may comprise components for carrying out any method disclosed herein.

[0019] In an aspect, there is provided a nucleic acid linker molecule, wherein one end is suitable for ligation to a DSB end and the other end is suitable for ligation to a sequencing adapter.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 illustrates the meaning of the term “DSB end”.

[0022] Figure 2 illustrates a species within an embodiment of a nucleic acid library as prepared herein. On the left is a region derived from the original DNA sample, and the location of the DSB end is illustrated. Next is a nucleic acid linker that has been ligated, in situ, to the DSB end. Finally, on the right, is a sequencing adapter with a bound motor protein that has been ligated, in vitro, to the linker.

[0023] Figure 3 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker via a single nucleotide overhang and the in vitro ligation of the sequencing adapter via a different single nucleotide overhang.

[0024] Figure 4 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker via a single nucleotide overhang and the in vitro ligation of the sequencing adapter via a sticky end.

[0025] Figure 5 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker via a single nucleotide overhang and the in vitro ligation of the sequencing adapter via a second linker comprising a sticky end.

[0026] Figure 6 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker, the end-repair and tailing of the linker, and the in vitro ligation of the sequencing adapter to the linker. The DSB ends can be identified in the reads due to the presence of the linker.

[0027] Figure 7 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker and the in vitro ligation of the sequencing adapter via a click-chemistry modifications.

[0028] Figure 8 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ modification of a DSB end to comprise a click modification and the ligation of the sequencing adapter via click-chemistry. Figure 9 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ modification of a DSB end to comprise a protective group and the ligation of the sequencing adapter to ends with a protective group.

[0029] Figure 10 illustrates an embodiment of the method of the first aspect. This embodiment involves the in situ ligation of a linker with a protective group at the unligated end and the in vitro ligation of the sequencing adapter to ends with a protective group.

[0030] Figure 11 shows an exemplary method as used in Example 3.

[0031] Figure 12 provides an example of a Hindlll recognition site (Hindlll recognition site 1) where breaks representing the expected 5’ overhang staggered cuts are observed.

[0032] Figure 13 provides an example of a Hindlll recognition site (Hindlll recognition site 2) where breaks representing the expected 5’ overhang staggered cuts are observed.

[0033] Figure 14 shows the recurrency of breaks at Hindlll recognition sites vs background. The darker colour represents non-Hindlll sites (background) and the lighter colour represents Hindlll recognition sites.

[0034] DETAILED DESCRIPTION

[0035] Current methods of preparing nucleic acid libraries containing labelled DSBs are not compatible with singlemolecule sequencing methods. It would be desirable to make use of single-molecule sequencing methods because they are associated with several potential advantages. For instance, such methods can generate long reads that are useful when mapping reads to low complexity regions of a genome. Long reads are also preferable for identifying genomic rearrangements, in addition, single-molecule sequencing methods can be used in a manner that does not cause the loss of epigenetic information and allows its direct identification.

[0036] To address these needs, the inventors provide herein methods for the preparation of nucleic acid libraries comprising labelled DSBs, wherein the nucleic acid libraries are compatible with single-molecule sequencing techniques. Sequencing of such libraries can generate reads that can be used to identify the location and / or frequency of DSBs within the original nucleic acid sample.

[0037] Thus, in a first aspect, there is provided a method of preparing a nucleic acid library for detecting DSBs in a nucleic acid sample, the method comprising labelling a DSB end with a sequencing adapter suitable for singlemolecule sequencing to generate the nucleic acid library.

[0038] As further discussed herein, in some embodiments the methods comprise: exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends in the nucleic acid sample; and exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate the nucleic acid library.

[0039] These embodiments may be expressed as: in situ modifying DSB ends in the nucleic acid sample; and attaching a sequencing adapter in vitro to the modified DSB ends in order to generate the nucleic acid library.

[0040] As will be elaborated upon, the in situ modification may be any allowing the attachment of a sequencing adapter to only the original DSB ends that were present in the sample, hence avoiding the attachment of the sequencing adapter to newly formed breaks (e.g. introduced during subsequent manipulation of the sample). In multiple examples herein, the modification is the attachment of a linker, for instance a nucleic acid adapter that can ligate to the DSB end and can be ligated by the sequencing adapter. In other embodiments, the modification protects or otherwise marks the DSB ends, such that the sequencing adapter can later be attached to only the protected or otherwise marked DSB ends. Illustrative embodiments include the in situ addition of an attachment moiety, such as a click modification, to DSB ends. Other illustrative embodiments include the in situ addition of a protective group, such as an exo-resistant nucleotide, to DSB ends.

[0041] The nucleic acid sample may be any suitable sample that may contain DSBs for detection. The nucleic acid sample may be a DNA sample. The DNA sample may be genomic DNA, organelle DNA, or cell-free DNA. The DNA sample may be from any suitable organism, including plants, mammals, and microorganisms. Particular embodiments include the detection of DSBs in genomic DNA, such as the DNA of crops or animals. An embodiment that is particularly relevant to medicine is the detection of DSBs in human genomic DNA. In any embodiments, the extraction and / or purification of the nucleic acids may be the extraction and / or purification of DNA.

[0042] The nucleic acid sample may be within, or may have been extracted from, a cell or a population of cells. The cell or population of cells may have been exposed to an agent or condition that can induce DSBs; that is suspected to be capable of inducing DSBs; or for which it is desirable to determine the presence, nature, number, location, and / or frequency of any induced DSBs. For example, the cell or cells may have been genetically modified. The cell or cells may have been exposed to a gene-editing technique. The cell or cells may have been exposed to an endonuclease, such as a CRISPR-Cas based system, a restriction enzyme, a TALEN, or the like. The genetic modification may be the knock-out, knock-down, overexpression, or other modification of an endogenous gene. The genetic modification may comprise the introduction of an exogenous nucleic acid sequence. An exogenous or modified protein may have been expressed within the cell or within an organism within which the cell is found. The agent or condition to which the cell or cells have been exposed may be nutrient starvation or excess, change in temperature, or any other change in conditions that might affect DSBs. The agent or condition may be the treatment of the cell or cells, such as the culturing conditions, conditions for purifying or isolating the cells, or the conditions for storing or shipping the cells. For instance, the agent or condition may be the conditions relating to freezing cells or thawing cells.

[0043] The DSBs may have been generated in the nucleic acid sample or may be generated in the nucleic acid sample by the conversion of a feature of interest into a DSB. The methods disclosed herein may therefore be used to identify such features by virtue of their conversion into DSBs.

[0044] Thus, the nucleic acid sample may contain one or more DSBs that have been converted from a feature of interest. For example, a lesion in one strand of double-stranded DNA may be enzymatically converted into a DSB, which can then be detected. In some examples, the lesion may be a single-strand break. In other examples, the feature of interest is a base change. For instance, the sample may contain base changes introduced by CRISPR / Cas base editing, such as a cytosine base editor or an adenosine base editor. Such edits may be converted into DSBs.

[0045] The nucleic acid sample may undergo or may have undergone a restriction enzyme digest, to induce breaks at features of interest. For instance, a restriction digest to introduce breaks at non-methylated sites.

[0046] In some embodiments, the nucleic acid sample has previously been exposed to an agent or conditions for the conversion of a feature of interest into a DSB. In some embodiments, the methods of the first aspect comprise exposing a nucleic acid sample to an agent or conditions for the conversion of a feature of interest into a DSB.

[0047] The DSBs may be used to identify sites of protein binding to a nucleic acid. For instance, a nucleic acid sample may be contacted with a protein-binding agent, such as an antibody, specific for a protein-of-interest, wherein the protein-of-interest is potentially bound to DNA. The DNA may be a sample that has been contacted with the protein-of-interest. The protein-binding agent may be directly or indirectly associated with a nuclease, hence forming a DSB at any site at which the protein of interest is bound. Any DSBs may then be detected by the methods disclosed herein. The methodology may be Cleavage Under Targets and Release Using Nuclease (CUT&RUN) methods. As such, the method of the first aspect may comprise: contacting the sample with a protein-of-interest and a nuclease capable of directly or indirectly associating with the protein-of-interest to form a DSB in nucleic acids to which the protein is bound.

[0048] The methods of first aspect may allow the detection of viral or bacterial insertion events or DNA damage caused by viral or bacterial insertion events. The methods can reveal sites of foreign DNA insertion, which can occur via a DSB intermediate structure.

[0049] The methods may allow the detection of contamination of a sample by any agent capable of causing DSBs.

[0050] The methods may be used to measure the stability of artificially assembled or synthetic genomes.

[0051] The methods may be useful for Next-Generation Risk Assessment (NGRA) in genetic toxicology. NGRA is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. DSBs are a direct measurement of genotoxic exposure and can be identified by the methods of the invention.

[0052] A nucleic acid library for detecting DSBs in a nucleic acid sample is one that can be sequenced to produce sequencing information that can be analysed to identify DSBs within the nucleic acid sample. In particular, the library will contain sequenceable nucleic acids that were originally directly adjacent to a DSB. For instance, one end of the sequenceable nucleic acid molecule is the site of the DSB in the original sample. The library may be enriched for sequenceable DSB-associated nucleic acids or the only sequenceable nucleic acids within the sample may be DSB-associated. The library may contain a mixture of sequenceable nucleic acids including both DSB- associated nucleic acids and non-D SB -associated nucleic acids, wherein the DSB-associated nucleic acids can be identified by analysis of sequence reads (e.g. by identification of a known sequence in an adapter / linker).

[0053] The preparation of a nucleic acid library involves obtaining a nucleic acid sample and modifying the sample such that it can be sequenced. As discussed herein, some of the steps of preparing the nucleic acid library may be performed before the nucleic acid sample is extracted from cells. Other steps may be performed after the nucleic acid sample has been extracted from the cells, for instance after DNA purification.

[0054] Many sequencing methods require the presence of a sequencing adapter ligated to a sample nucleic acid to render the nucleic acid sequenceable. The methods of the first aspect involve labelling a DSB end with a suitable adapter in a manner that allows the labelled sample nucleic acids to be sequenced. A “DSB end” as used herein is a terminus of a nucleic acid molecule that has been generated by a DSB. This is illustrated in Figure 1. The sequencing adapter may be attached directly or indirectly to the DSB end, and further embodiments are discussed herein.

[0055] The sequencing adapter may be double-stranded. The sequencing adapter may be attached to the 5’ terminus at a DSB end and / or to the 3 ’ terminus at a DSB end. The sequencing adapter may be attached to both strands of a DSB end (i.e. both the 3’ and the 5’ termini).

[0056] As discussed herein, there may be a linker in-between the sequencing adapter and the DSB end. The linker may be double stranded. The linker may be a double-stranded nucleic acid molecule designed to act as an adapter to which a second adapter may ligate. The linker may be attached to the 5’ terminus at a DSB end and / or to the 3’ terminus at a DSB end. The linker may be attached to both strands of a DSB end (i.e. both the 3’ and the 5’ termini). The sequencing adapter may be attached to the linker. The linker may be double stranded, and the sequencing adapter may be attached to both strands of the linker. The sequencing adapter may be attached to the 5 ’ terminus of the linker and / or to the 3 ’ terminus of the linker. The sequencing adapter may be attached to both strands of the linker ( / . e. both the 3 ’ and the 5 ’ termini) . Further embodiments of methods of labelling a D SB end with a sequencing adapter are provided herein. In some embodiments, the sequencing adapter comprises two strands of which one is the strand that will interact with the sequencing technology. For instance, the sequencing adapter may comprise a strand that is ligated to the strand of the nucleic acid that is eventually sequenced. In embodiments where the nucleic acid library is suitable for nanopore sequencing, this may be the strand that can bind or has bound a motor protein. In these embodiments, the permanent attachment of this strand is the most important and attachment of the non-sequenced strand is optional. In some embodiments, the sequencing adapter comprises a motor protein bound to a first strand, and the 5’ end of the first strand should be fused (directly or indirectly) to the DSB end. In examples, the sequencing adapter comprises a motor protein bound to a first strand, and the 5 ’ end of the first strand is fused directly to a strand of a linker, and the strand of the linker is fused directly to the DSB end.

[0057] A sequencing adapter suitable for single-molecule sequencing is a sequencing adapter that enables the nucleic acid molecule to which it is attached, directly or indirectly (e.g. via a linker), to be sequenced by a method of singlemolecule sequencing. As used herein “single-molecule sequencing” refers to techniques where sequencing information is obtained directly from a nucleic acid strand. Single-molecule sequencing techniques generate a read from one template molecule, whereas non-single-molecule sequencing techniques generate a read from multiple, or a cluster, of template molecules. In some embodiments, the nucleic acid strand may be an original strand of a nucleic acid molecule that was present in the sample. Thus the sequencing method may derive sequencing information from an original molecule rather than from a copy or copies of the nucleic acid molecules. In other embodiments, the original nucleic acid molecule is RNA, such as mRNA, which is converted into a cDNA molecule and the cDNA molecule is directly sequenced. In such examples, a single break in RNA may be converted into a DSB in DNA. The sequencing adapters that are suitable for use of the first aspect may be adapters that do not require the library to be amplified, e.g. the library does not need to be subject to PCR, prior to sequencing.

[0058] In some instances, the single-molecule sequencing technique does not require the creation of a copy of the sample nucleic acid, and hence may preserve the epigenetic information. The nucleic acid library may therefore be suitable for sequencing and for the direct detection of epigenetic modifications. Thus, the method of the first aspect may be a method of preparing a nucleic acid library for detecting DSBs and epigenetic modifications. The epigenetic modifications may be methylation of DNA. The modifications may be CpG methylation. Illustrative examples of modifications that may be detected are 5mC, 5hmC, and 6mA.

[0059] The sequencing adapter may be suitable for use with a method of long-read sequencing. A long read may be a read that is greater than Ikb in length.

[0060] The sequencing adapter may comprise an index sequence.

[0061] Examples of single-molecule sequencing include techniques that involve the measurement of a signal that is affected differently by each base within the single molecule, hence allowing the determination of the sequence. In an example, a nucleic acid molecule is passed across a partition, electrical current is measured as the nucleic acid molecule traverses the partition, and the sequence is identified due to changes in the electrical current that are characteristic of each base. The partition may be an electro-resistant membrane containing pores or channels through which the nucleic acid molecule can pass. The pores or channels may be a protein nanopore set into a membrane.

[0062] A more specific example is known in the art as nanopore sequencing. Nanopore sequencing involves passing a nucleic acid molecule through a protein nanopore set into an electro-resistant membrane and measuring the electrical current during this process. Examples of flow cells for nanopore sequencing are the Oxford Nanopore R9.4.1 MinlON or GridlON flow cell or R9.4.1 PromethlON flow cell, the Oxford Nanopore R10.4.1 MinlON or GridlON flow cell or R10.4.1 PromethlON flow cell, or the R10.4.1 Flongle flow cell. The sequencing adapter may be suitable for use with MinlON™, GridlON™, or PromethlON™ technologies.

[0063] Sequencing adapters suitable for nanopore sequencing are known in the art and are commercially available. Nanopore sequencing techniques may require the sequencing adapter to be bound to a motor protein that is compatible with the nanopore. Thus, the method of the first aspect may comprise labelling a DSB end with a sequencing adapter suitable for nanopore sequencing. The sequencing adapter may comprise a binding site that is suitable for the binding of a motor protein, and so the sequencing adapter may be one to which a motor protein suitable for nanopore sequencing can be attached. The method may comprise labelling a DSB end with a sequencing adapter suitable for nanopore sequencing that is bound to a motor protein. And so the motor protein and the sequencing adapter may already be complexed at the time of labelling the DSB end, e.g. the motor protein and the sequencing adapter may already be complexed at the time of attaching a sequencing adapter directly or via a linker to the DSB end.

[0064] In some embodiments, changes may be made to the other end of the nucleic acid molecule (i.e. the end to which the sequencing adapter is not ligated). For instance, another adapter may be ligated to the other end. In some examples, the adapter at the other end is a U-shaped adapter or a hairpin adapter. This adapter at the other end may allow duplex sequencing. And so both strands may be sequenced as a single molecule. In examples involving a nanopore, a hairpin adapter may allow both strands to pass through the pore as a single molecule.

[0065] It can be desirable to label only DSB ends that were present before further processing of the nucleic acid sample; this ensures that additional DSBs introduced into the sample by library preparation are not labelled, sequenced, and mistakenly identified as DSBs present within the original sample. However, this can be challenging when preparing a nucleic acid library for nanopore sequencing because the motor protein may be altered or damaged during further steps. For instance, it is desirable to attach the sequencing adapter to the DSB end in situ (and so before purification of nucleic acids from cells) and prior to further fragmentation. But the in situ attachment of a sequencing adapter complexed to a motor protein to a DSB end means that the motor protein will be subjected to the conditions required for the purification of nucleic acids from cells and potentially to conditions required for fragmentation of the nucleic acid sample.

[0066] To address the aforementioned issues, the inventors provide methods herein that involve the attachment of a linker to the DSB end. In embodiments comprising more than one linker this linker is referred to as the “first linker” herein. The linker may be attached to the DSB end in in situ and so may be attached intracellularly. This means that the linker is attached to the DSB ends before further processing that might introduce additional DSBs.

[0067] The in situ addition of the linker to the DSB ends may be considered the in situ modification of DSB ends such that later (e.g. after DNA purification) the sequencing adapter can be attached to the modified DSB ends.

[0068] The linker may be a double-stranded nucleic acid molecule designed to act as an adapter to which a second adapter may ligate. The linker may be a nucleic acid molecule and at least 5 base pairs (bp), at least 10 bp, at least 15 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 75 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, or at least 500 bp in length.

[0069] Some methods of sequencing, such as nanopore sequencing, are less accurate at the start of a read and increase in accuracy during the read. The linker may be or may comprise a nucleic acid strand of sufficient length such that the low accuracy portion of the read is the linker and the higher accuracy portion is the nucleic acid from the sample. In some embodiments, the method of sequencing are low accuracy for around 100 to 130 bps, and so the linker may be at least about 100 bp or at least about 130 bp in length. The linker may be about 100 bp to about 500 bp in length. The linker may be about 100 bp to about 300 bp in length. The linker may be about 100 bp to about 200 bp in length. The linker may be about 100 bp, about 200 bp, about 300 bp, about 400 bp or about 500 bp in length. The linker may be any range between these numbers.

[0070] In any of the embodiments disclosed herein, the linker may comprise an index sequence. The linker may comprise one or more primer binding sites. For instance, a primer binding site for targeted PCR or strand synthesis. The linker may comprise one or more unique molecule identifiers (UMIs). The linker may comprise one or more base modifications for physical pull down enrichment (e.g. biotin).

[0071] The method of the first aspect may therefore comprise obtaining a cell, wherein the cell comprises the nucleic acid sample for analysis. The method of the first aspect may comprise obtaining a population of cells, wherein the population of cells comprises the nucleic acid sample for analysis. The cell or population of cells may already have been exposed to an agent or conditions for the induction or potential induction of DSBs, as discussed herein. In embodiments comprising the conversion or potential conversion of features of interest into DSBs, the cells may be treated or have been treated so as to induce this conversion. The cell or cells may be fixed. In some examples, the cell or cells are immobilised or in suspension. The cell or cells may be permeabilised. The cell or cells may first be fixed and then permeabilised, or may be subjected to a combined fixation and permeabilization step. The fixation and permeabilization conditions are such that the cells are suitable for subsequent steps of the method. For instance, the fixation and permeabilization may render the cells suitable for end-repairing and / or tailing DNA breaks within the cell. The fixation and permeabilization may render the cells suitable for attachment of an exogenous nucleic acid adapter to DNA breaks within the cell.

[0072] Thus, the method of the first aspect may comprise: obtaining a cell and fixing and permeabilising the cell. In some embodiments, the method of the first aspect comprises: obtaining a population of cells and fixing and permeabilising the population of cells.

[0073] In some embodiments, after fixation and permeabilization, the linker is introduced to the cell or cells under conditions suitable for the attachment of the first linker to any DSB ends. For instance, the linker may be a nucleic acid molecule and may be introduced under ligation conditions.

[0074] The ligation may comprise the annealing of one or more terminal unpaired bases at the DSB end to complementary one or more terminal unpaired bases comprised by the linker. The ligation may comprise the annealing of an overhang at the DSB end to a complementary overhang on the nucleic acid linker. The ligation may comprise the annealing of an underhang at the DSB end to a complementary underhang on the nucleic acid linker. To achieve this, prior to the ligation of the linker, the DSB ends may be end repaired and the overhang or underhang may be added. Thus, the method of the first aspect may comprise end-repairing of DSB ends in the sample and the adding of a nucleotide overhang or underhang. Methods for end-repair are known in the art and comprise blunting. Methods for blunting are known in the art and include endo-nuclease blunting or exo-nuclease blunting.

[0075] The one or more terminal unpaired bases may be a single nucleotide. Thus, the method of the first aspect may comprise tailing the DSB end, for instance A-tailing, T-tailing, C-tailing, or G-tailing. In a particular embodiment the DSB end is G-tailed and the linker has a C-tail. In another embodiment, the DSB end is A-tailed and the first linker has a T-tail. Methods for tailing are also known in the art.

[0076] The one or more terminal unpaired bases may be a series of the same base. For instance, a poly(A) tail, poly(T) tail, poly(C) tail, or poly(G) tail. In an example, a poly(T) tail may be added by TdT. In some instances, a mixture of regular and terminating bases (e.g. ddTTP) is used to control the length of the tail.

[0077] The end-repair / blunting and tailing may be performed as separate steps, or as a single step. After attachment of the linker, the nucleic acids may be purified. For instance, where the attachment takes place intracellularly, the nucleic acids may be extracted from the cell or population of cells after the attachment of the linker. In some embodiments, DNA is purified from the cell or cells after the linker has been attached. The extraction may comprise, or be subsequent to, cell lysis. Thus, the methods of the first aspect may comprise: cell lysis, DNA extraction, and DNA purification. These steps may be consecutive or combined.

[0078] The methods may comprise a step of fragmenting or shearing the nucleic acids after extraction. The nucleic acids may be fragmented or sheared so as to be of a suitable size for the intended downstream sequencing. If the intended downstream sequencing is a method of obtaining long reads, the fragmentation step should be such that suitably sized fragments are obtained. For example, the fragments may be over 1 kb. Fragmentation may take place by methods known in the art, such as by sonication.

[0079] In other examples, a fragmentation step is not used. This may lead to libraries comprising long nucleic acids (e.g. 100 kb or more). Alternatively, the nucleic acids may be gently sheared by pipette mixing or light vortexing, e.g. to generate libraries around 20kb average size.

[0080] The method may comprise a step of removing nucleic acid fragments that are below a certain size threshold. In addition, or alternatively, the method may comprise a step of removing nucleic acid fragments that are above a certain size threshold. For instance, this step may be used to remove excess linker. If it is possible for linker concatemers to form, this step may remove said concatemers. The step of selecting certain fragment sizes may be performed by conventional means, such as by the use of solid-phase reversible immobilization (SPRI) beads. Other means of clean-up include the use of precipitation, nanobind disks, other beads. The techniques used for clean-up may be chosen to suit the nature of the library, e.g. some clean-up techniques are suitable for ultra-long library preparation methods.

[0081] In a particular embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, and extracting nucleic acids from the cell or cells.

[0082] In a particular embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, and extracting nucleic acids from the cell or cells.

[0083] In another embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, end-repairing and tailing DSB ends within the cell or cells, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker can anneal to the DSB end via a complementary tail, and extracting nucleic acids from the cell or cells. In another embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, end-repairing and tailing DSB ends within the cell or cells, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, wherein the linker anneals to the DSB end via a complementary tail, and extracting nucleic acids from the cell or cells.

[0084] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0085] After the attachment of the linker, the method may comprise the attachment of a sequencing adapter to the linker. This may take place in vitro. And so this step may take place after the purification of the nucleic acids, for instance after extraction of the nucleic acids from a cell or population of cells. The method may comprise a step of DNA purification after the linker has been attached to any DSB ends.

[0086] The attachment of the sequencing adapter may be ligation. The ligation may comprise the annealing of one or more terminal unpaired bases comprised by the linker to complementary one or more terminal unpaired bases comprised by the sequencing adapter. The ligation may comprise the annealing of an overhang of the linker to a complementary overhang of the sequencing adapter. The ligation may comprise the annealing of an underhang of the linker to a complementary underhang of the sequencing adapter.

[0087] In a particular embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, extracting nucleic acids from the cell or cells, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to the linker.

[0088] In a particular embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, extracting nucleic acids from the cell or cells, and attaching the sequencing adapter to the linker.

[0089] In another embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, end-repairing and tailing DSB ends within the cell or cells, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker can anneal to the DSB end via a complementary tail, extracting nucleic acids from the cell or cells, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to the linker, wherein the sequencing adapter can anneal to the linker via complementary one or more terminal unpaired bases.

[0090] In another embodiment, the method of the first aspect comprises: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, end-repairing and tailing DSB ends within the cell or cells, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, wherein the linker anneals to the DSB end via a complementary tail, extracting nucleic acids from the cell or cells; attaching the sequencing adapter to the linker, wherein the sequencing adapter anneals to the linker via complementary one or more terminal unpaired bases.

[0091] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0092] The one or more terminal unpaired bases may be a single nucleotide. In a particular embodiment, the linker has an A-tail and the sequencing adapter has a T-tail. In another embodiment, the linker has a G-tail and the sequencing adapter has a C-tail. The one or more terminal unpaired bases may be a series of the same base. For instance, a poly(A) tail, poly(T) tail, poly(C) tail, or poly(G) tail.

[0093] Some embodiments comprise the ligation of the linker to the DSB end comprising the annealing of single nucleotide overhangs and the ligation of the sequencing adapter to the linker comprising the annealing of single nucleotide overhangs. In such embodiments, the linker-DSB-end ligation may involve the annealing of a different base pair to the linker-sequencing-adapter ligation. In examples, the ligation of the DSB end and the linker may involve annealing a G-tail and a C-tail, whereas the ligation of the linker and the sequencing adapter may involve the annealing of an A-tail and a T-tail. In other examples, the ligation of the DSB end and the linker may involve annealing an A-tail and a T-tail, whereas the ligation of the linker and the sequencing adapter may involve the annealing of a G-tail and a C-tail.

[0094] Thus, prior to any ligation steps, the linker may have one or more terminal unpaired bases at one end that are suitable for ligation to the DSB end and one or more terminal unpaired bases at the other end that are suitable for ligation of the sequencing adapter. The linker may have a single nucleotide overhang at a first end and a different single nucleotide overhang at the other end. The linker may have a C-tail at one end and an A-tail at the other end. The linker may have a G-tail at one end and a T-tail at the other end.

[0095] A particular embodiment is illustrated in Figure 3. In this figure, DSBs are end-repaired and G-tailed in situ. A linker is then ligated to the DSBs and the ligation comprises annealing of a C-tail to the G-tail. The linker comprises an A overhang at the unligated end. After linker ligation, the nucleic acids are extracted and optionally fragmented. A sequencing adapter is then ligated to the linker and the ligation comprises annealing of a T-tail to the A-tail. The nucleic acid library may proceed to sequencing.

[0096] In other embodiments, the linker may comprise a sticky -end and the ligation of the sequencing adapter may comprise annealing to the sticky-end. A sticky -end, as used herein, is an overhang or underhang of more than one unpaired nucleotide to which a complementary sticky-end may anneal. In some instances, the linker comprises a restriction site that can be cut with a restriction enzyme to reveal the sticky end. In other instances, the linker comprises the sticky end in a form that is ready for annealing and does not require cutting as a part of the methods of the first aspect.

[0097] In some examples, prior to any ligation steps, the linker may have one or more terminal unpaired bases at one end that are suitable for ligation to the DSB end and a sticky -end at the other end that is suitable for ligation of the sequencing adapter. The linker may have a single nucleotide overhang at a first end and a sticky -end at the other end. The linker may have a T-tail at one end and a sticky -end at the other end.

[0098] A particular embodiment is illustrated in Figure 4. In this figure, DSBs are end-repaired and A-tailed in situ. A linker is then ligated to the DSBs and the ligation comprises annealing of a T-tail to the A-tail. The linker comprises a sticky -end (a non-limiting embodiment is illustrated) at the unligated end. After linker ligation, the nucleic acids are extracted and optionally fragmented. A sequencing adapter is then ligated to the linker and the ligation comprises annealing to the sticky -end. The nucleic acid library may proceed to sequencing.

[0099] In some embodiments, the sequencing adapter comprises a sticky -end that is complementary to the sticky -end of the linker. However, it may be convenient to attach the sticky -end to the sequencing adapter via a second linker (the linker attached to the DSB end being the first linker). Thus, the method may comprise ligating a second linker to the sequencing adapter, wherein the second linker comprises a sticky -end that is complementary to the sticky -end of the linker. An advantage of embodiments comprising the second linker is that they may be more easily applicable to commercially available sequencing adapters.

[0100] The second linker may comprise features as disclosed for the first linker. For instance, the second linker may be a double-stranded nucleic acid molecule. The second linker may comprise an index sequence. The second linker may comprise one or more primer binding sites. For instance, a primer binding site for targeted PCR or strand synthesis. The second linker may comprise one or more UMIs. The second linker may comprise one or more base modifications for physical pull down enrichment (e.g. biotin).

[0101] A particular embodiment is illustrated in Figure 5. In this figure, DSBs are end-repaired and A-tailed in situ. A first linker is then ligated to the DSBs and the ligation comprises annealing of a T-tail to the A-tail. The first linker comprises a sticky -end at the unligated end. After first linker ligation, the nucleic acids are extracted and optionally fragmented. A second linker has been ligated to the sequencing adapter, and the second linker provides a sticky-end that is complementary to the sticky -end of the first linker. The method then comprises the ligation of the sequencing adapter to the first linker and the ligation comprises annealing to the sticky -ends of the first and second linkers. The nucleic acid library may proceed to sequencing.

[0102] In some embodiments, the linker is treated to add one or more terminal unpaired bases after it has been attached to the DSB end. This adds one or more terminal unpaired bases that are suitable for the ligation of the sequencing adapter. This step may take place in situ or in vitro. In particular, the step takes place in vitro as discussed below.

[0103] In other embodiments, the linker (first linker) is attached to the DSB end by any of the methods disclosed herein. In particular, the linker may be attached to the DSB end by in situ ligation. The nucleic acids are then purified (e.g. extracted from cells) and optionally fragmented. The sample is then treated to add one or more terminal unpaired bases to the linker, wherein the one or more terminal unpaired bases are suitable for the ligation of the sequencing adapter. For instance, the sample may be end-repaired and tailed to add a single nucleotide overhang to the linker. The sample may be end-repaired and tailed to add a series of the same base. For instance, a poly(A) tail, poly(T) tail, poly(C) tail, or poly(G) tail.

[0104] In these embodiments, a tail may have been added to the DSB end for linker ligation and the same tail may be later added to the linker for sequencing adapter ligation. For instance, the DSB end may have been A-tailed for linker ligation and, subsequently, the unligated end of the linker may be A-tailed for ligation of the sequencing adapter. Both ligations may comprise single nucleotide A-tails.

[0105] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, extracting nucleic acids from the cell or cells, end-repairing and tailing any linkers present, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to the linker.

[0106] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, extracting nucleic acids from the cell or cells, end-repairing and tailing the linker, and attaching the sequencing adapter to the linker.

[0107] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0108] In these embodiments, nucleic acids that are not associated with a DSB may, or will, be rendered suitable for ligation by the sequencing adapter. In this situation, DSB -associated nucleic acids may be identified due to the presence of the linker. For instance, the linker may comprise a known nucleic acid sequence and so reads from DSB -associated nucleic acids may be identified as they will comprise the known sequence from within the linker.

[0109] A particular embodiment is illustrated in Figure 6. In this figure, DSBs are end-repaired and tailed in situ. A linker is then ligated to the DSBs. After linker ligation, the nucleic acids are extracted and optionally fragmented. The method then comprises the end-repair and A-tailing of the sample, this adds an A-tail to the ligated linker as well as at other DNA termini (e.g. as introduced by fragmentation). Next, the sequencing adapter is provided under conditions suitable for ligation, which will result in the sequencing adapter being ligated to the linkers and also to other end-repaired- A-tailed termini. The nucleic acid library may proceed to sequencing. Reads from DSB -associated nucleic acids can be identified by in silico analysis to identify reads from linker-associated nucleic acids.

[0110] The attachment of the linker and / or sequencing adapter to the target nucleic acid molecule can be by any suitable means. Several embodiments discussed herein operate according to conventional ligation techniques, but the invention is not limited to only traditional ligation. For instance, the target molecule for attachment may comprise a first attachment moiety and the nucleic acid to be attached may comprise a second attachment moiety. The first and the second attachment moieties are such that they can interact to attach the two nucleic acids. The attachment may happen because the first and the second attachment moieties react to form a bond attaching the moieties and hence the nucleic acids. The bond may be a chemical bond. The first and the second attachment moieties may be used to attach the sequencing adapter to the linker (already attached to the DSB end). In other embodiments, the first and the second attachment moieties are for attachment of the sequencing adapter directly to the DSB end (and so the DSB end itself is modified to comprise an attachment moiety). The in situ modification of a DSB end to add an atachment moiety may be considered the in situ modification of DSB ends such that later (e.g. after DNA purification) the sequencing adapter can be atached to the modified DSB ends.

[0111] As discussed herein, the sequencing adapter and / or linker may be double-stranded nucleic acid molecules. In such embodiments, the attachment moieties should be present on the strand-to-be-sequenced. For instance, in embodiments where the sequencing adapter is suitable for nanopore sequencing and can bind or has bound a motor protein, the strand of the adapter that will interact with the nanopore for sequencing should comprise the atachment moiety, such that the relevant strand of the sequencing adapter is atached (directly or indirectly) to the DSB end. The atachment of the non-sequenced strand is optional.

[0112] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for atachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a first attachment moiety at the end not atached to the DSB end, extracting nucleic acids from the cell or cells, providing the sequencing adapter to the extracted nucleic acids under conditions suitable for atachment of the sequencing adapter to the linker, wherein the sequencing adapter comprises a second attachment moiety that can bind to or react with the first atachment moiety.

[0113] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, wherein the linker comprises a first atachment moiety at the end not atached to the DSB end, extracting nucleic acids from the cell or cells, attaching the sequencing adapter to the linker, wherein the sequencing adapter comprises a second atachment moiety that can bind to or react with the first atachment moiety.

[0114] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, exposing the cell or cells to conditions for adding a first atachment moiety to DSB ends, extracting nucleic acids from the cell or cells, providing the sequencing adapter to the extracted nucleic acids under conditions suitable for atachment of the sequencing adapter to the linker, wherein the sequencing adapter comprises a second attachment moiety that can bind to or react with the first atachment moiety.

[0115] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, adding a first atachment moiety to DSB ends, extracting nucleic acids from the cell or cells, attaching the sequencing adapter to the DSB end, wherein the sequencing adapter comprises a second attachment moiety that can bind to or react with the first attachment moiety.

[0116] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0117] As an example, the first and second attachment moieties may operate by click-chemistry. The first and second attachment moieties may be click modifications that chemically bond to each other by a click-chemistry reaction. The first and second click-chemistry modifications may be used to connect any two nucleic acids as disclosed herein.

[0118] Azide-alkyne cycloaddition methods (e.g. copper-catalysed azide-alkyne cycloaddition (CuAAC)) and strain- promoted azide-alkyne cycloaddition (SPAAC)) are the most common click chemistry -based approaches used for oligonucleotide ligation. However, alternatives such as the Thiol-Ene Reaction, Thiol-Michael Addition, Diels- Alder Reactions, Tetrazine IEDDA, and Oxime Ligation are also suitable.

[0119] In particular, strain-promoted azide-alkyne cycloaddition (SPAAC) has strong bioorthogonality (i.e. specificity in biological systems) and absence of toxicity associated with the need for a copper catalyst. Azide- and strained alkyne groups for nucleotide labelling are readily available and may be used for 5’ or 3’ labelling.

[0120] The click chemistry may be azide-alkyne cycloaddition (AAC). The click modifications can, therefore, comprise or be an azide group and an alkyne moiety. The azide and the alkyne moieties react spontaneously and highly specifically to form covalent bonds. Hence, these moieties can be used as attachment moieties to fuse two nucleic acids.

[0121] The click chemistry may be “copper-free click chemistry. This does not require the addition of copper and so is not toxic to live cells. In particular embodiments, the click modifications comprise or are an azide moiety and a ‘strained’ -alkyne such as DBCO.

[0122] In some embodiments, the first linker (the linker for direct attachment to the DSB end) comprises the first click modification. The click modification may be present at the end of the linker that is not for attachment to the DSB end. In such embodiments, the sequencing adapter may comprise the second click modification, and so the first and second click modifications allow for the sequencing adapter to be attached to the linker. As discussed, the strand-to-be-sequenced should be fused (directly or indirectly) to the sequencing adapter. Thus, in some embodiments, the linker comprises a first click modification at the 3 ’ terminus at the end not for attachment to the DSB end, and the sequencing adapter comprises a second click modification at the 5’ terminus of the end of the sequencing adapter for attachment (see Figure 7 for an illustrative embodiment).

[0123] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a first click modification at the 3 ’ terminus of the end not attached to the DSB end, extracting nucleic acids from the cell or cells, providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to the linker, wherein the sequencing adapter comprises a second click modification, which can react with the first click modification, at the 5’ terminus of the end to be ligated to the linker. The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, wherein the linker comprises a first click modification at the 3 ’ terminus of the end not attached to the DSB end, extracting nucleic acids from the cell or cells, attaching the sequencing adapter to the linker, wherein the sequencing adapter comprises a second click modification, which can react with the first click modification, at the 5’ terminus of the end to be attached to the linker.

[0124] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0125] A particular embodiment is illustrated in Figure 7. In this figure, DSBs are end-repaired and A-tailed in situ. A linker is then ligated to the DSBs and the ligation comprises annealing of a T-tail to the A-tail. The linker comprises a first click modification at the 3’ terminus of the unligated end. After linker ligation, the nucleic acids are extracted and optionally fragmented. A sequencing adapter is then ligated to the linker by click-chemistry, wherein the sequencing adapter comprises a second click modification (which is reactive with the first) at the 5’ terminus of the end for ligation. The nucleic acid library may proceed to sequencing.

[0126] In other embodiments, the DSB end comprises the first click modification and the sequencing adapter comprises the second click modification, and so the first and second click modifications allow for the sequencing adapter to be directly attached to the DSB end. As discussed, the strand-to-be-sequenced should be fused to the sequencing adapter. Thus, in some embodiments, the DSB end comprises a first click modification at the 3 ’ terminus and the sequencing adapter comprises a second click modification at the 5’ terminus of the end of the sequencing adapter for attachment (see Figure 8 for an illustrative embodiment).

[0127] The in situ addition of the click modification (or other attachment moiety) to the DSB ends may be considered the in situ modification of DSB ends such that later (e.g. after DNA purification) the sequencing adapter can be attached to the modified DSB ends.

[0128] The DSB end does not naturally comprise the first click modification, and so the method of the first aspect can comprise the addition of a first click modification to a DSB end. As discussed, where the sequencing adapter comprises one strand that will interact with the sequencing technology, this is the strand that should be fused (the fusing of the other strand is optional). In some embodiments, this means that the method of the first aspect comprises the addition of a first click modification to a 3 ’ terminus at a DSB end. Methods for adding a click modification to a DSB end include extending the 3 ’ terminus with TdT in the presence of DBCO / Azide dATP.

[0129] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, exposing the cell or cells to conditions for the addition of a first click modification to DSB ends, extracting nucleic acids from the cell or cells, providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to the DSB end, wherein the sequencing adapter comprises a second click modification that can bind to or react with the first click modification.

[0130] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, adding a first click modification to DSB ends, extracting nucleic acids from the cell or cells, attaching the sequencing adapter to the DSB ends, wherein the sequencing adapter comprises a second click modification that can bind to or react with the first click modification.

[0131] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0132] A particular embodiment is illustrated in Figure 8. In this figure, the 3 ’ termini of DSB ends are extended in situ with TdT in the presence of DBCO / Azide dATP. The nucleic acids are then extracted and optionally fragmented. A sequencing adapter comprising a second click modification reactive with the click modification of the DSB end is then attached to the DSB -end. The nucleic acid library may proceed to sequencing.

[0133] In other embodiments, the nucleic acid sample may be treated to protect any DSB ends. In some embodiments, the nucleic acid sample is treated to add a protective group to the DSB end. This step may take place in situ. This step may take place intracellularly and so before the nucleic acid sample has been extracted from a cell or from cells. The protection of the DSB ends is such that the sample can be treated to ensure that attachment may take place only to the DSB ends, even if further breaks have been introduced after the protection of the DSB ends. For instance, the DSBs may be protected intracellularly before DNA purification and optionally fragmentation, and any attachment steps subsequently will only attach to the protected DSB ends and not to any newly introduced breaks. The sequencing adapter may then be directly attached to the protected DSB ends, and this step may take place in vitro.

[0134] The in situ protection of the DSB ends may be considered the in situ modification of DSB ends such that later (e.g. after DNA purification) the sequencing adapter can be attached to the modified DSB ends.

[0135] In an embodiment, the DSB ends are protected in situ by the addition of an exo-resistant residue. For instance, the exo-resistant residue may be an exo-resistant adenosine such as an inverted dA. After further processing, which may introduce new breaks, the sample can be treated to render breaks without an exo-resistant residue to be unsuitable for ligation or attachment. For instance, the sample may be treated to blunt unprotected ends or to extend unprotected ends with irrelevant nucleotides. After this treatment, which leaves only the original DSB ends as available for attachment or ligation, the sample may be exposed to the sequencing adapter under conditions suitable for attachment or ligation.

[0136] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, exposing the cell or cells to conditions to protect DSB ends, extracting nucleic acids from the cell or cells, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to protected DSB ends.

[0137] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, protecting DSB ends, extracting nucleic acids from the cell or cells, and attaching the sequencing adapter to protected DSB ends.

[0138] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, exposing the cell or cells to conditions to protect DSB ends by the addition of an exo-resistant reside, extracting nucleic acids from the cell or cells and optionally fragmenting the nucleic acids, exposing the extracted nucleic acids to conditions to blunt or extend unprotected breaks, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for attachment of the sequencing adapter to protected DSB ends.

[0139] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, protecting DSB ends by the addition of an exo-resistant reside, extracting nucleic acids from the cell or cells and optionally fragmenting the nucleic acids, treating the sample to blunt or extend unprotected breaks, and attaching the sequencing adapter to protected DSB ends.

[0140] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0141] A particular embodiment is illustrated in Figure 9. In this figure, DSBs are end-repaired and A-tailed in situ with an exo-resistant adenosine. After treatment, the nucleic acids are extracted and optionally fragmented. The sample is then treated to blunt non-protected ends or to extend non-protected ends with residues that are not adenosine. A sequencing adapter is then ligated to the DSB end and the ligation comprises annealing of a T-tail to the A-tail. The nucleic acid library may proceed to sequencing.

[0142] In another embodiment, a linker that comprises a protective group is attached to the DSB ends. The protective group may be located at the end of the linker that is not attached to the DSB end. The attachment of the linker may take place intracellularly and so before the nucleic acid sample has been extracted from a cell or from cells. The protection of the unattached end of the linker is such that the sample can be treated to ensure that attachment may take place only to the linker, even if further breaks have been introduced after the attachment (e.g. during purification and / or fragmentation of DNA). The sequencing adapter may then be attached to the protected linker, and this step may take place in vitro.

[0143] In an embodiment, the linker comprises a protected 5’ phosphate. The 5’ phosphate may be located at the end of the linker that is not for attachment to the DSB end. Prior to attachment of the sequencing adapter, the nucleic acid sample may be exposed to conditions for the dephosphorylation of any unprotected 5’ phosphates, thus rendering unligatable breaks introduced by, for instance, extraction and / or fragmentation.

[0144] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a protected 5’ phosphate at the end not attached to the DSB end, extracting nucleic acids from the cell or cells, exposing the sample to conditions to dephosphorylate any unprotected 5’ phosphates, and providing the sequencing adapter under conditions suitable for ligation to the linker.

[0145] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a nucleic acid adapter (an embodiment of the linker) to DSB ends within the cell or cells, wherein the linker comprises a protected 5’ phosphate at the end not attached to the DSB end, extracting nucleic acids from the cell or cells, dephosphorylating any unprotected 5’ phosphates, and ligating the sequencing adapter to the linker.

[0146] In the methods above, the steps are performed in order but there may be additional steps that intervene the recited steps. In addition, there may be additional steps prior to or after the above steps.

[0147] A particular embodiment is illustrated in Figure 10. In this figure, DSBs are end-repaired and A-tailed in situ. A linker is then ligated to the DSBs and the ligation comprises annealing of a T-tail to the A-tail. The linker comprises a protected 5’ phosphate at the unligated end. After linker ligation, the nucleic acids are extracted and optionally fragmented. Any unprotected 5’ phosphates are then dephosphorylated. The sample is then A-tailed in vitro. A sequencing adapter is then ligated to the linker and the ligation comprises annealing of a T-tail to the A- tail. The nucleic acid library may proceed to sequencing.

[0148] In some embodiments, a linker is attached to a DSB end in situ according to any of the examples discussed herein, and the linker comprises a purification moiety. The purification moiety may be any moiety that can be bound by a binding moiety to allow purification, enrichment, or pull down of a nucleic acid molecule to which the purification moiety is attached. The methods may therefore comprise the step of contacting the nucleic acid sample with binding moiety and isolating nucleic acid molecules associated with a purification moiety bound to the binding moiety.

[0149] The purification moiety and binding moiety may be a ligand-receptor pair or a receptor-ligand pair. In an example, the pair is biotin-streptavidin.

[0150] The binding moiety may be immobilised to a substrate. The substrate may be ahead. The substrate may allow the separation of molecules bound, directly or indirectly, to the substrate from other molecules in a sample.

[0151] In embodiments comprising enrichment, the attachment of the linker to the DSB end and the attachment of sequencing adapter to the linker may be according to any techniques disclosed herein.

[0152] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, enriching for nucleic acid molecules associated with the purification moiety, and providing the sequencing adapter under conditions suitable for attachment to the enriched nucleic acid molecules.

[0153] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, enriching for nucleic acid molecules associated with the purification moiety, and attaching the sequencing adapter to the linker.

[0154] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, extracting nucleic acids from the cell or cells, contacting the extracted nucleic acids with a binding moiety, enriching for nucleic acid molecules associated with purification moieties bound to the binding moiety, and providing the sequencing adapter under conditions suitable for attachment to the enriched nucleic acid molecules.

[0155] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, extracting nucleic acids from the cell or cells, contacting the extracted nucleic acids with a binding moiety, enriching for nucleic acid molecules associated with purification moieties bound to the binding moiety, and attaching the sequencing adapter to the linker.

[0156] In some examples, the enriching for nucleic acid molecules associated with purification moieties may be performed after the sequencing adapter has been attached. And so the enrichment may take place on the nucleic acid library to enrich the nucleic acid library for the desired species. In these embodiments, the linker (first linker) may comprise the purification moiety. Alternatively, or in addition, the second linker (where present) and / or the sequencing adapter may comprise a purification moiety.

[0157] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, providing the sequencing adapter under conditions suitable for attachment to the linker, optionally wherein the sequencing adapter is attached to the linker via a second linker, and enriching for nucleic acid molecules associated with a purification moiety associated with the linker, second linker, and / or the sequencing adapter.

[0158] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, providing the sequencing adapter under conditions suitable for attachment to the linker, and enriching for nucleic acid molecules associated with the purification moiety.

[0159] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, attaching the sequencing adapter to the linker, and enriching for nucleic acid molecules associated with the purification moiety.

[0160] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, providing a nucleic acid adapter (an embodiment of the linker) to the cell or cells under conditions suitable for attachment of the linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, extracting nucleic acid molecules from the cell or cells, providing the sequencing adapter under conditions suitable for attachment to the extracted nucleic acid molecules to form a nucleic acid library, contacting the nucleic acid library with a binding moiety, and enriching for nucleic acid molecules associated with purification moieties bound to the binding moiety.

[0161] The method of the first aspect may comprise: obtaining a cell or population of cells, wherein the cell or population of cells has optionally been exposed to an agent or condition for which it is desired to determine the effect on the induction of DSBs or wherein a feature of interest has been converted into a DSB, attaching a linker to any DSB ends within the cell or cells, wherein the linker comprises a purification moiety, extracting nucleic acids from the cell or cells, attaching the sequencing adapter to the linker of the extracted nucleic acids to form a nucleic acid library, contacting the nucleic acid library with a binding moiety, and enriching for nucleic acid molecules associated with purification moieties bound to the binding moiety. In any embodiments, after attachment of the sequencing adapter the nucleic acid library may be further processed before sequencing. For instance, there may be further purification steps. There may be steps to remove nucleic acid fragments that are below a certain size threshold. For instance, this step may be used to remove excess sequencing adapter. If it is possible for concatemers to form, this step may remove said concatemers. The step of selecting certain fragment sizes may be performed by conventional means, such as by the use of solid-phase reversible immobilization (SPRI) beads.

[0162] The methods of the first aspect may comprise sequencing the nucleic acid library. The sequencing is singlemolecule sequencing, as disclosed herein. The sequencing may result in reads that are greater than 250bp, greater than 500bp, greater than 750bp, greater than Ikb, greater than lOkb, greater than 50kb, greater than lOOkb, or greater than 1Mb. The sequencing may be long-read sequencing. The sequencing may involve the measurement of a signal that is affected differently by each base within the single molecule, hence allowing the determination of the sequence. The sequencing may involve passing the nucleic acid molecule across a partition, measuring the electrical current as the nucleic acid molecule traverses the partition, and identifying the sequence due to changes in the electrical current that are characteristic of each base. The partition may be an electro-resistant membrane containing pores or channels through which the nucleic acid molecule can pass. In a specific example, the sequencing is nanopore sequencing (discussed further herein). The sequencing may comprise adaptive sampling, for instance adaptive sampling that uses either the base-called sequence or the raw signal (current). Adaptive sampling allows the enrichment of relevant molecules in the library during the sequencing process.

[0163] Sequencing produces sequence reads and the method may comprise aligning the reads to a reference sequence. The reference sequence will reflect the original nucleic acid sample, for instance if the original sample was genomic DNA, the reference sequence will be a reference genome for the relevant organism. In an embodiment, the original nucleic acid sample is DNA from human cells and the reference sequence is a reference human genome.

[0164] After alignment of the reads to the reference sequence, the method may comprise identification of the location of DSBs within the nucleic acid sample. The reads are derived from D SB -associated nucleic acids, allowing this identification to be made. In embodiments comprising the attachment of linkers to the DSB end, the method may comprise the identification of a portion of the sequence of the linker within the reads, and this known sequence may be used to identify the first base adjacent to the DSB.

[0165] The methods of the first aspect may comprise detecting the presence / absence of epigenetic modifications in the nucleic acid library. The epigenetic modifications may be methylation of DNA. The modifications may be CpG methylation. Illustrative examples of modifications that may be detected are 5mC, 5hmC, and 6mA.

[0166] In a second aspect, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising sequencing a nucleic acid library prepared by a method of the first aspect.

[0167] In an embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends in the nucleic acid sample; exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate the nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0168] In an embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: in situ modifying DSB ends in the nucleic acid sample; attaching a sequencing adapter in vitro to the modified DSB ends in order to generate the nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0169] In an embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ attachment of a first linker to DSB ends in the nucleic acid sample; exposing the nucleic acid sample to conditions suitable for in vitro attachment of a sequencing adapter to the first linker in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0170] In another embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: attaching in situ a first linker to DSB ends in the nucleic acid sample; attaching in vitro a sequencing adapter to the first linker in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

[0171] All of the features disclosed for the first aspect are relevant to the second aspect. And so the nucleic acid sample many be any sample, and may be treated or may have been treated, as disclosed for the first aspect.

[0172] The method of library preparation may be any method of the first aspect. Including any technique disclosed herein of attaching a linker to a DSB end and / or attaching a sequencing adapter to a linker, or any technique disclosed herein of attaching a sequencing adapter to a DSB end. Also included in the second aspect are the methods involving attachment moieties, protective groups, and / or purification moieties.

[0173] The sequencing may be any method of sequencing as discussed for the first aspect. In particular examples, the sequencing is nanopore sequencing.

[0174] Sequencing produces sequence reads and the methods of the second aspect may comprise aligning the reads to a reference sequence. The reference sequence will reflect the original nucleic acid sample, for instance if the original sample was genomic DNA, the reference sequence will be a reference genome for the relevant organism. In an embodiment, the original nucleic acid sample is DNA from human cells and the reference sequence is a reference human genome. After alignment of the reads to the reference sequence, the methods may comprise identification of the location of DSBs within the nucleic acid sample. The reads are derived from DSB -associated nucleic acids, allowing this identification to be made. As mentioned, in embodiments comprising a linker the known sequence of the linker may be identified in the read to locate the first base adjacent to the DSB.

[0175] Thus, the methods may comprise sequencing the nucleic acid library using single-molecule sequencing to generate sequence reads, and then aligning the sequence reads to a reference sequence in order to detect and identify the location of any DSBs within the nucleic acid sample.

[0176] The methods of the second aspect may comprise detecting the presence / absence of epigenetic modifications in the nucleic acid library. The epigenetic modifications may be as discussed for the first aspect.

[0177] In another embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: providing a first linker to a cell under conditions suitable for the attachment of the first linker to any DSB ends within the cell, extracting nucleic acids from the cell, exposing the extracted nucleic acids to conditions suitable for the attachment of a sequencing adapter to the first linker, and sequencing the nucleic acids using single-molecule sequencing. In another embodiment, there is provided a method of detecting DSBs in a nucleic acid sample, the method comprising: attaching a first linker to DSB ends within a cell, extracting nucleic acids from the cell, attaching a sequencing adapter to the first linker, and sequencing the nucleic acids using single-molecule sequencing.

[0178] In a third aspect, there is provided a kit suitable for carrying out a method of the first aspect or the second aspect.

[0179] In an embodiment, there is provided a kit for preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the kit comprising a linker (first linker) as disclosed herein.

[0180] In an embodiment, there is provided a kit for preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the kit comprising a linker and a sequencing adapter suitable for single-molecule sequencing.

[0181] The linker may be any as discussed for the first aspect or the fourth aspect. The sequencing adapter may be any as discussed for the first aspect. The linker may be a first linker as discussed for the first aspect or fourth aspect and the features discussed, such as length etc, are also relevant to the third aspect.

[0182] The linker has a first end and a second end. The first end may comprise one or more terminal unpaired bases.

[0183] The first end may comprise an overhang or an underhang. The nature of the terminal unpaired bases, overhang, or underhang may be any as disclosed for the first aspect. The one or more terminal unpaired bases may be a single nucleotide.

[0184] The second end of the linker may comprise one or more terminal unpaired bases. The second end of the linker may comprise an overhang or an underhang. The nature of the terminal unpaired bases, overhang, or underhang may be any as disclosed for the first aspect. The one or more terminal unpaired bases may be different from the one or more terminal unpaired bases of the first end. The one or more terminal unpaired bases may be a single nucleotide or may be a sticky end.

[0185] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a different single nucleotide overhang at the second end. In another embodiment, the linker comprises a single nucleotide overhang at the first end and a sticky-end at the second end.

[0186] The second end of the linker may comprise a first attachment moiety. The nature of the first attachment moiety may be as disclosed for the first aspect. For instance, the first attachment moiety may be a click modification.

[0187] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a first attachment moiety at the second end.

[0188] The second end of the linker may comprise a protective group. The nature of the protective group may be as disclosed for the first aspect. For instance, the protective group may be a protected 5’ phosphate.

[0189] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a protective group at the second end.

[0190] The sequencing adapter may comprise one or more terminal unpaired bases. The sequencing adapter may comprise an overhang or underhang. The nature of the terminal unpaired bases, overhang, or underhang may be any as disclosed for the first aspect. The terminal unpaired bases, overhang, or underhang may be complementary to the terminal unpaired bases, overhang, or underhang of the second end of the linker. The one or more terminal unpaired bases may be a single nucleotide or may be a sticky end.

[0191] The sequencing adapter may comprise a second attachment moiety. The nature of the second attachment moiety may be as disclosed for the first aspect. For instance, the second attachment moiety may be a click modification. The first attachment moiety and the second attachment moiety can bind to each other or can react to form a bond attaching the first and the second attachment moieties.

[0192] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises one or more terminal unpaired bases at the first end and different one or more terminal unpaired bases at the second end, and a sequencing adapter comprising one or more terminal impaired bases that are complementary to the one or more terminal unpaired bases at the second end of the linker.

[0193] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises an overhang or underhang at the first end and a different overhang or underhang at the second end, and a sequencing adapter comprising an overhang or underhang that is complementary to the overhang or underhang at the second end of the linker.

[0194] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises a single nucleotide overhang at the first end and a different single nucleotide overhang at the second end, and a sequencing adapter comprising a single nucleotide overhang that is complementary to the single nucleotide overhang at the second end of the linker.

[0195] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises a single nucleotide overhang at the first end and a sticky -end at the second end, and a sequencing adapter comprising a sticky -end that is complementary to the sticky -end at the second end of the linker.

[0196] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises a single nucleotide overhang at the first end and a first attachment moiety at the second end, and a sequencing adapter comprising a second attachment moiety, wherein the first attachment moiety and the second attachment moiety can bind to each other or can react to form a bond attaching the first and the second attachment moieties. Optionally, the first and second attachment moieties are click modifications.

[0197] In an embodiment, the kit comprises a linker that has a first end and a second end, wherein the linker comprises a single nucleotide overhang at the first end and a protective group at the second end, and a sequencing adapter. Optionally wherein the protective group is a protected 5’ phosphate.

[0198] In some embodiments, the kit comprises a second linker.

[0199] In an embodiment, there is provided a kit for preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the kit comprising a first linker and a second linker. The first linker may be any as disclosed herein and, in particular, may comprise a sticky -end at the opposite end (the second end) to the end for attachment to the DSB end. The second linker may comprise a sticky -end that is complementary to the sticky - end at the second end of the first linker.

[0200] In a particular embodiment, the first linker has a first end and a second end, wherein the first linker comprises a single nucleotide overhang at the first end and a sticky-end at the second end; and the second linker comprises a first end and a second end, wherein the second linker comprises at the first end a sticky-end that is complementary to the sticky-end of the first linker, and the second linker comprises a single nucleotide overhang at the second end.

[0201] The kit may comprise the first linker, the second linker, and the sequencing adapter. In a particular embodiment, the first linker has a first end and a second end, wherein the first linker comprises a single nucleotide overhang at the first end and a sticky-end at the second end; the second linker comprises a first end and a second end, wherein the second linker comprises at the first end a sticky-end that is complementary to the sticky-end of the first linker, and the second linker comprises a single nucleotide overhang at the second end; and the sequencing adapter comprises a single nucleotide overhang that is complementary to the single nucleotide overhang at the second end of the second linker.

[0202] The kit may comprise a fixation buffer. The fixation buffer is one that may be applied to a cell or population of cells to fix the nucleic acids therein.

[0203] The kit may comprise a permeabilization buffer. The permeabilization buffer is one that may be applied to a cell or population of cells to permeabilise the cell membrane such that nucleic acid molecules, such as the linkers disclosed herein, may enter the cell and interact with the nucleic acids therein.

[0204] The kit may comprise a buffer capable of both fixation and permeabilization. A fixation and permeabilization buffer is one that may be applied to a cell or population of cells to simultaneously fix the nucleic acids therein and permeabilise the cell membrane such that nucleic acid molecules, such as the linkers disclosed herein, may enter the cell and interact with the nucleic acids therein.

[0205] The kit may comprise one or more end-repair enzymes and a suitable end-repair reaction buffer in an end-repair enzyme mix. The end repair enzyme mix may be suitable to be applied to a cell or population of cells such that DSB overhangs and underhangs are converted into blunt-ends suitable for nucleotide -tailing in situ.

[0206] The kit may comprise one or more nucleotide -tailing enzymes and a suitable tailing reaction buffer in a tailing enzyme mix. The tailing enzyme mix may be suitable to be applied to a cell or population of cells to add a non- templated nucleotide or nucleotides to the 3’ termini of blunt-ended DSBs in situ. The non-templated nucleotides may be resistant to exonuclease activity.

[0207] The kit may comprise an end-repair / tailing reaction mix for end-repair and nucleotide-tailing in a single step. The end-repair / tailing reaction mix is one that may be applied to a cell or population of cells to convert DSB ends into blunt-ends suitable for nucleotide-tailing and to ligate non-templated nucleotides to the 3’ termini of blunt-ended DNA in a single step.

[0208] The kit may comprise one or more linkers, adapters, ligation enzymes, and a suitable ligation reaction buffer in a ligation reaction mix. The ligation reaction mix may be applied to a cell or population of cells to anneal and ligate the nucleic acid molecules via complementary base pairing of terminal unpaired nucleotide or nucleotides.

[0209] The kit may comprise the necessary components for end repair, nucleotide-tailing, and ligation in the same reaction vessel. The components may be applied to a cell or population of cells to convert DSBs into blunt-ends, to add non-templated nucleotides to the 3’ termini of the DSB blunt-ends, and to anneal and ligate nucleic acid molecules via complementary base pairing of terminal unpaired nucleotides.

[0210] The kit may comprise lysis reagents. The lysis reagents may be applied to a cell or population of cells to disrupt the cell membrane such that a lysate is generated comprising the nucleic acids and other cellular contents therein. The lysis reagents may comprise physical reagents, enzymatic reagents, chemical reagents, or combinations thereof.

[0211] The kit may comprise a lysate clearing reagents. The lysate clearing reagents may be applied to a lysate to remove unwanted cellular contents prior to DNA purification.

[0212] The kit may comprise purification reagents. The reagents may be for the purification of DNA.

[0213] The kit may comprise the necessary components for lysis, lysis clearing, DNA purification, or a combination thereof in a single step.

[0214] The kit may comprise DNA fragmentation reagents.

[0215] The kit may contain reagents suitable for removing nucleic acid fragments that are above or below a certain size threshold. These reagents may be or may comprise solid-phase reversible immobilization (SPRI) beads.

[0216] The kit may comprise nucleic acid dephosphorylation reagents.

[0217] The kit may contain reagents for adding click modifications to nucleic acid strands. The kit may comprise reagents for adding any of the click modifications disclosed herein. For instance, the kit may contain reagents for adding a click modification for attachment via azide-alkyne cycloaddition. In an example, the kit contains reagents for extending a 3 ’ terminus with TdT in the presence of DBCO / Azide dATP. So the kit may comprise a suitable buffer containing DBCO / Azide dATP and may optionally contain TdT.

[0218] In a fourth aspect, there is provided a linker as described for the first aspect, second aspect, or third aspect.

[0219] The linker may be any first linker or second linker as disclosed herein. The features as discussed for the first aspect, such as length etc, are also relevant to the fourth aspect.

[0220] In a particular embodiment, the linker is a double-stranded nucleic acid adapter molecule, designed to attach or ligate to a DSB end in a DNA sample and designed to be attached or ligated by a second adapter. The linker may be a double-stranded linear nucleic acid molecule. The linker may comprise an index or barcode sequence. The linker may comprise one or more primer binding sites. For instance, a primer binding site for targeted PCR or strand synthesis. The linker may comprise one or more UMIs. The linker may comprise one or more base modifications for physical pull down enrichment (e.g. biotin).

[0221] The linker may comprise a known sequence in known proximity to the end that will attach to the DSB end. The linker may comprise a nucleic acid strand of sufficient length such that, when sequencing nucleic acids that include a fusion of “sample-to-linker-to-sequencing-adapter”, the low accuracy portion of reads is the linker and the higher accuracy portion is the nucleic acid from the sample.

[0222] The linker may be a nucleic acid molecule and at least 5 base pairs (bp), at least 10 bp, at least 15 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 75 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 300 bp, at least 400 bp, or at least 500 bp in length. The linker may be about 100 bp to about 500 bp in length. The linker may be about 100 bp to about 300 bp in length. The linker may be about 100 bp to about 200 bp in length. The linker may be about 100 bp, about 200 bp, about 300 bp, about 400 bp or about 500 bp in length. The linker may be any range between these numbers.

[0223] The linker has a first end and a second end. The first end may comprise one or more terminal unpaired bases.

[0224] The first end may comprise an overhang or an underhang. The nature of the terminal unpaired bases, overhang, or underhang may be any as disclosed for the first aspect or third aspect. The one or more terminal unpaired bases may be a single nucleotide.

[0225] The second end of the linker may comprise one or more terminal unpaired bases. The second end of the linker may comprise an overhang or an underhang. The nature of the terminal unpaired bases, overhang, or underhang may be any as disclosed for the first aspect or third aspect. The terminal unpaired bases may be different from the terminal unpaired bases of the first end. The one or more terminal unpaired bases may be a single nucleotide or may be a sticky end.

[0226] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a different single nucleotide overhang at the second end. In another embodiment, the linker comprises a single nucleotide overhang at the first end and a sticky-end at the second end.

[0227] The second end of the linker may comprise a first attachment moiety. The nature of the first attachment moiety may be as disclosed for the first aspect. For instance, the first attachment moiety may be a click modification.

[0228] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a first attachment moiety at the second end.

[0229] The second end of the linker may comprise a protective group. The nature of the protective group may be as disclosed for the first aspect. For instance, the protective group may be a protected 5’ phosphate.

[0230] In an embodiment, the linker comprises a single nucleotide overhang at the first end and a protective group at the second end.

[0231] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0232] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0233] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the Examples, which are not intended to limit the invention in any way.

[0234] EXAMPLES

[0235] Example 1- Summary of some advantages of some aspects of the invention

[0236] 1. It specifically labels the double-stranded breaks at the earliest possible stage in the process. This label can be used to ‘enrich’ for double-stranded breaks in multiple ways (see examples herein). 2. The linker, when present, serves as a marker; enabling further breaks to be made during the preparation process and optionally prepared as sequencing library alongside the labelled breaks. These new breaks can easily be differentiated from original DSBs via the presence / absence of the linker sequence.

[0237] 3. The linker also serves as an ‘anchor-point’ for later ligation steps (i.e. for ligation of a sequencing adapter). This ‘anchor-point’ can be modified in various ways to adjust ligation specificity and / or efficiency. Options include: a single-base overhang / underhang; a multi-base ‘sticky -end’ for high ligation specificity and efficiency, click-chemistry modifications for simple and specific rapid attachment.

[0238] 4. The linker sequence also serves as a ‘buffer’ for sequencing by providing an initial expected sequence prior to the start of the broken end to maximise read-quality and precise capture of the break site.

[0239] 5. The conserved sequence of the linker oligo also enables rapid identification of labelled vs unlabelled breaks via the raw sequencing signal (current) or during / after base-calling, enabling enrichment of data via selective sequencing (e.g. rapid adaptive sequencing). Alternatively, the conserved sequence of the linker would allow post-hoc bioinformatic filtering of the data to find these sequences which marked breaks that existed in situ.

[0240] 6. The linker is robust to extraction / manipulation steps.

[0241] Example 2 - Illustrative non-limiting protocol

[0242] Cell work:

[0243] 1. According to experimental questions cells treated or otherwise

[0244] 2. Cells immobilised on plate eg Poly-D-lysine, lamin coated plates, by fixative (or fixed in solution) eg by poly -formaldehyde (PF A) or ethanol

[0245] 3. Wash with PB S to remove fixative

[0246] In situ steps:

[0247] 4. Permeabilize cells

[0248] 5. PBS wash

[0249] 6. Buffer wash

[0250] 7. (Optional) convert feature of interest to double strand breaks (DSB) eg nick or base change

[0251] 8. Buffer wash

[0252] 9. End repair DSB break sites

[0253] 10. (Depending on strategy) A-tail (optional exo-resistant A) or G-tail DSB sites or extend 3 ’ ends with TdT and DBCO or Azide dATP

[0254] 11. Buffer wash

[0255] 12. (Depending on strategy) Ligate linker with AT ligation, GC ligation or click chemistry depending on strategy

[0256] 13. (Depending on strategy) Washes to remove excess linker

[0257] Extraction of gDNA steps:

[0258] 14. Extract gDNA and purify

[0259] 15. (Optional) Fragment or shear gDNA to desired size range

[0260] 16. (Optional) Size select gDNA of desired size range

[0261] 17. (Depending on strategy) Experimental enrichment of gDNA with labelled linker (at DSB locations) eg biotin-streptavidin pull-down

[0262] Library preparation steps: 18. (Depending on strategy) End repair linker, A-tail

[0263] 19. (Depending on strategy) Dephosphorylate ends and A-tail

[0264] 20. (Depending on strategy) Ligate second linker to sequencing adapter and purify

[0265] 21. (Depending on strategy) Blunt unprotected ends or extend with non-A

[0266] 22. Ligate sequencing adapter to prepared gDNA via AT ligation or sticky overhang ligation or click chemistry

[0267] 23. (Depending on strategy) Experimental enrichment of libraries with labelled linker (at DSB locations), second linker or adapter eg biotin-streptavidin pull-down

[0268] 24. Purify sequencing libraries

[0269] Sequencing steps:

[0270] 25. Prepare sequencer and flow cell

[0271] 26. Load sequencing library onto flow cell

[0272] 27. Sequence

[0273] 28. (Depending on strategy) On-device enrichment for libraries with linker (at DSB locations) due to only these sites being converted into functional sequencing libraries

[0274] 29. (Optional) On-device enrichment for libraries with linker (at DSB locations) by adaptive sampling

[0275] 30. (Optional) Enrich for reads with presence of linker (at DSB locations) bioinformatically post-sequencing

[0276] 31. (Optional) Determine epigenetic DNA modifications from sequencing reads using appropriate software

[0277] Example 3 - Evidence for break labelline

[0278] An experiment was performed to test whether breaks at Hindlll recognition sites could be detected using a method disclosed herein. The workflow is depicted in Figure 11. In summary, a population of cells was treated with Hindlll to digest nucleic acids within the cells. This was followed by in situ end-repair and A-tailing. A lOObp linker with a T overhang was then ligated, in situ, to A-tailed nucleic acids. The linker included a sticky end at the non-ligated end. SPRI beads were used to purify DNA from the population of cells and g-tube fragmentation was performed. A sequencing adapter for Oxford Nanopore sequencing was ligated to the linker via the sticky end of the linker. PCR was not performed. The resultant library was then sequenced using a MinlON sequencer via on- device enrichment.

[0279] Analysis of the sequencing data revealed that breaks were observed at the expected Hindlll sites in genomic DNA. Figures 12 and 13 which are two examples of Hindlll sites where breaks representing an expected 5’ overhang staggered cut were observed.

[0280] The frequency of high recurrency breaks at Hindlll sites was highly enriched (Figure 14) when compared to endogenous background sites. Across all Hindlll sites, breaks occurred at >350-fold higher than expected by random chance.

[0281] 77% of the reads from this experiment were derived from breaks labelled with the adapter. The Hindlll breaks had the expected structure.

[0282] In summary, the methods disclosed herein can be used to detect nucleic acid breaks.

Claims

CLAIMS1. A method of preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the method comprising labelling a DSB end with a sequencing adapter suitable for single-molecule sequencing to generate the nucleic acid library.

2. The method of claim 1, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends in the nucleic acid sample; and exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate the nucleic acid library.

3. The method of claim 1 or claim 2, wherein the nucleic acid library is suitable for the direct detection of epigenetic modifications.

4. The method of any one of claims 1 to 3, the method comprising: attaching a first linker to the DSB end.

5. The method of claim 4, wherein the attachment of the first linker to the DSB end takes place in situ.

6. The method of claim 4 or claim 5, wherein a cell or population of cells comprises the nucleic acid sample, and the attachment of the first linker takes place intracellularly.

7. The method of any one of claims 4 to 6, wherein after attachment of the first linker to the DSB end, the nucleic acid sample is extracted from cells.

8. The method of any one of claims 1 to 7, the method comprising: obtaining a cell, fixing and permeabilising the cell, providing a first linker to the cell under conditions suitable for the attachment of the first linker to any DSB ends within the cell, and extracting nucleic acids from the cell.

9. The method of any one of claims 4 to 8, wherein the first linker is a double-stranded nucleic acid molecule and the first linker is attached to DSB ends using a first ligation method.

10. The method of claim 9, wherein the DSB end comprises one or more terminal unpaired bases and the first linker comprises one or more terminal unpaired bases that are complementary, and the first ligation method comprises the annealing of the terminal unpaired bases of the first linker to the terminal unpaired bases of the DSB end.

11. The method of claim 9 or claim 10, wherein the DSB end comprises an overhang or underhang and the first linker comprises a complementary overhang or underhang, and the first ligation method comprises the binding of the first linker to the DSB end via the overhangs.

12. The method of any one of claims 9 to 11, wherein the DSB end comprises a single nucleotide overhang and the first linker comprises a complementary single nucleotide overhang, and the first ligation method comprises the binding of the first linker to the DSB end via the overhangs.

13. The method of any one of claims 1 to 12, the method comprising: end-repairing any DSB ends within a cell and adding a single nucleotide overhang (tailing) to repaired DSB ends, providing a first linker to the cell, wherein the first linker comprises a single nucleotide overhang that is complementary to the single nucleotide overhang of the DSB ends, and providing conditions for the ligation of the first linker to DSB ends, and extracting nucleic acids from the cell.

14. The method of any one of claims 10 to 13, wherein the DSB end has a G tail and the first linker has a C-tail, or the DSB end has an A-tail and the first linker has a T-tail.

15. The method of any one of claims 4 to 14, further comprising: attaching the sequencing adapter to the first linker.

16. The method of claim 15, wherein the attachment of the sequencing adapter to the first linker takes place in vitro.

17. The method of claim 15 or claim 16, wherein the attachment of the sequencing adapter the first linker takes place after extraction of the nucleic acid sample from cells.

18. The method of any one of claims 1 to 17, the method comprising: providing a first linker to a cell under conditions suitable for the attachment of the first linker to any DSB ends within the cell, extracting nucleic acids from the cell, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for the attachment of the sequencing adapter to the first linker.

19. The method of any one of claims 15 to 18, wherein the sequencing adapter is attached to the first linker using a second ligation method.

20. The method of claim 19, wherein the first and / or second ligation method comprises complementary base pairing.

21. The method of claim 20, wherein the first linker comprises one or more terminal unpaired bases and the sequencing adapter comprises one or more terminal unpaired bases that are complementary, and the second ligation method comprises the annealing of the terminal unpaired bases of the first linker to the terminal unpaired bases of the sequencing adapter.

22. The method of claim 20 or claim 21, wherein the first linker comprises an overhang or underhang and the sequencing adapter comprises a complementary overhang or underhang, and the second ligation method comprises the binding of the sequencing adapter to the first linker via the overhangs or underhangs.

23. The method of claim 20 or claim 21, wherein the first linker comprises a single nucleotide overhang and the sequencing adapter comprises a complementary single nucleotide overhang, and the second ligation method comprises the binding of the sequencing adapter to the first linker via the overhangs.

24. The method of any one of claims 1 to 23, the method comprising: end-repairing any DSB ends within a cell and adding a single nucleotide overhang (tailing) to repaired DSB ends,providing a first linker to the cell, wherein at a first end the first linker comprises a single nucleotide overhang that is complementary to the single nucleotide overhang of the DSB ends, and at a second end the first linker comprises a single nucleotide overhang that is complementary to a single nucleotide overhang of the sequencing adapter, and exposing the nucleic acid sample to conditions for the ligation of the first linker to the DSB ends, extracting nucleic acids from the cell, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for the ligation of the sequencing adapter to the first linker.

25. The method of claim 24, wherein the single nucleotide overhang at the first end of the first linker is different from the single nucleotide overhang at the second end of the first linker.

26. The method of any one of claims 21 to 25 wherein the DSB end has a G-tail, the first end of the first linker has a C-tail, the second end of the first linker has an A-tail, and the sequencing adapter has a T-tail.

27. The method of any one of claims 3 to 26, wherein the first linker comprises a sticky -end.

28. The method of claim 27, wherein at a first end the first linker comprises a single nucleotide overhang that is complementary to a single nucleotide overhang of the DSB ends, and at a second end the first linker comprises a sticky -end.

29. The method of claim 27 or claim 28, wherein the sequencing adapter comprises a sticky -end that is complementary to the sticky -end of the first linker.

30. The method of any one of claims 1 to 29, the method comprising: end-repairing any DSB ends within a cell and adding a single nucleotide overhang (tailing) to repaired DSB ends, providing a first linker to the cell, wherein at a first end the first linker comprises a single nucleotide overhang that is complementary to the single nucleotide overhang of the DSB ends, and at a second end the first linker comprises a sticky -end complementary to a sticky -end of the sequencing adapter, and exposing the nucleic acid sample to conditions for the ligation of the first linker to the DSB ends, extracting nucleic acids from the cell, and providing the sequencing adapter to the extracted nucleic acids under conditions suitable for the ligation of the sequencing adapter to the first linker.

31. The method of any one of claim 29 or claim 30, wherein the sticky-end of the sequencing adapter is attached to the sequencing adapter via a second linker.

32. The method of claim 31, wherein a second linker is ligated to the sequencing adapter, wherein the second linker has a first end that comprises a sticky-end complementary to the sticky-end of the first linker, and a second end that is ligated to the sequencing adapter.

33. The method of any one of claims 4 to 20, comprising, after attachment of the first linker to the DSB end and after extraction of the nucleic acid sample from cells: end-repairing the first linker, and adding one or more terminal unpaired bases to the repaired first linker.

34. The method of any one of claims 4 to 20, comprising, after attachment of the first linker to the DSB end and after extraction of the nucleic acid sample from cells: end-repairing the first linker, and adding a single nucleotide overhang to the repaired first linker.

35. The method of claim 33 or claim 34, wherein the sequencing adapter comprises one or more terminal unpaired bases that are complementary to the one or more terminal unpaired bases of the first linker, and the second ligation method comprises the binding of the sequencing adapter to the first linker via the terminal unpaired bases; or the sequencing adapter comprises an overhang that is complementary to the overhang of the first linker, and the second ligation method comprises the binding of the sequencing adapter to the first linker via the overhangs.

36. The method of any one of claims 1 to 35, the method comprising: providing conditions for the attachment of a first linker to DSB ends within a cell, extracting nucleic acids from the cell, end-repairing the attached first linker and adding one or more terminal unpaired bases to the repaired first linker, wherein the one or more terminal unpaired bases are complementary to terminal unpaired bases of the sequencing adapter, and providing the sequencing adapter under conditions suitable for the attachment of the sequencing adapter to the first linker.

37. The method of any one of claims 4 to 36, wherein the first linker or the DSB end comprises a first attachment moiety.

38. The method of claim 36, wherein at a first end the first linker comprises a single nucleotide overhang that is complementary to a single nucleotide overhang of the DSB end, and at a second end the first linker comprises a first attachment moiety.

39. The method of any one of claims 4 to 37, comprising adding a first attachment moiety to DSB ends in the nucleic acid sample.

40. The method of any one of claims 37 to 39, wherein the sequencing adapter comprises a second attachment moiety, wherein the first and the second attachment moieties can bind to each other or can react to form a bond attaching the first and the second attachment moieties.

41. The method of claim 40, wherein the first and second attachment moieties are click modifications; optionally wherein first and second attachment moieties bind due to a chemical reaction that is azide-alkyne cycloaddition; and / or wherein attachment moieties are azide and DBCO.

42. The method of any one of claims 1 to 38 and 40 to 41, the method comprising: attaching a first linker to DSB ends within a cell, wherein the first linker comprises a first attachment moiety at the unattached end, and attaching the sequencing adapter to the first linker, wherein the sequencing adapter comprises a second attachment moiety, wherein the first and the second attachment moieties bind to each other or react to form a bond attaching the first and the second attachment moieties.

43. The method of any one of claims 1 to 37 and 39 to 41, the method comprising: attaching a first attachment moiety to DSB ends within a cell, and attaching the sequencing adapter to the DSB ends, wherein the sequencing adapter comprises a second attachment moiety, wherein the first and the second attachment moieties bind to each other or react to form a bond attaching the first and the second attachment moieties.

44. The method of any one of claims 1 to 43, comprising treating the nucleic acid sample to protect any DSB ends.

45. The method of claim 44, wherein the DSB ends are protected by the addition of an exo-resistant residue.

46. The method of claim 45, wherein the exo-resistant residue is an exo-resistant adenosine.

47. The method of claim 46, wherein the exo-resistant adenosine is an inverted dA.

48. The method of any one of claims 44 to 47, comprising treating unprotected ends such that they are unsuitable for ligation or attachment, wherein the treating unprotected ends comprises blunting and / or extension with irrelevant nucleotides.

49. The method of any one of claims 4 to 48, comprising treating the first linker to protect the unattached end of the first linker.

50. The method of any one of claims 4 to 48, wherein the unattached end of the first linker comprises a protective group.

51. The method of claim 49 or claim 50, wherein the unattached end of the first linker is protected by a protected 5’ phosphate.

52. The method of any one of claims 44 to 51, comprising treating unprotected ends such that they are unsuitable for ligation or attachment, wherein the treating unprotected ends comprises dephosphorylating 5’ phosphate groups.

53. The method of any one of claims 44 to 52, comprising treating unprotected ends such that they are unsuitable for ligation or attachment.

54. The method of any one of claims 44 to 48 or 53, comprising attaching the sequencing adapter to protected DSB ends.

55. The method of claim 54, wherein the DSB ends are protected by an exo-resistant residue, and the sequencing adapter comprises an overhang complementary to said residue.

56. The method of any one of claims 44 to 48 or 53 to 55, wherein the protection of DSB ends takes place in situ and / or intracellularly.

57. The method of any one of claims 48, 52, or 53, wherein the treatment of unprotected ends takes place in vitro and / or after nucleic acid extraction from cells.

58. The method of any one of claims 1 to 57, the method comprising: treating the nucleic acid sample to protect any DSB ends, extracting nucleic acids from the cell, treating unprotected ends within the extracted nucleic acids such that they are unsuitable for ligation or attachment, and providing the sequencing adapter under conditions suitable for the attachment of the sequencing adapter to the DSB ends.

59. The method of any one of claims 1 to 57, the method comprising: providing a first linker a cell under conditions suitable for the attachment of the first linker to DSB ends within the cell, wherein the first linker comprises a protective group at the unattached end, extracting nucleic acids from the cell, treating unprotected ends such that they are unsuitable for ligation or attachment, and providing the sequencing adapter under conditions suitable for the attachment of the sequencing adapter to the first linker.

60. The method of any one of claims 4 to 59, wherein the first linker, second linker (where present), and / or sequencing adapter comprises a purification moiety.

61. The method of claim 60, comprising enriching for nucleic acid molecules associated with the purification moiety.

62. The method of claim 60 or claim 61, comprising contacting the nucleic acid sample with a binding moiety that can bind to the purification moiety.

63. The method of any one of claims 4 to 62, the method comprising: attaching a first linker to DSB ends within a cell, wherein the first linker comprises a purification moiety, enriching for nucleic acid molecules associated with the purification moiety.

64. The method of any preceding claim, wherein the sequencing adapter is suitable for a method of long-read sequencing.

65. The method of any preceding claim, wherein the sequencing adapter is suitable for nanopore sequencing.

66. The method of any preceding claim, wherein a motor protein is bound to the sequencing adapter or wherein the sequencing adapter has a binding site for a motor protein.

67. The method of claim 66, wherein the motor protein is compatible with a nanopore for sequencing.

68. The method of any preceding claim, comprising sequencing nucleic acids with an attached sequencing adapter.

69. The method of claim 68, comprising alignment to a reference sequence of sequence reads generated by the sequencing.

70. The method of claim 69, comprising identification of the location of DSBs within the nucleic acid sample.

71. A method of detecting DSBs in a nucleic acid sample, the method comprising:exposing the nucleic acid sample to conditions suitable for in situ modification of DSB ends; exposing the nucleic acid sample to in vitro conditions suitable for attachment of a sequencing adapter to the modified DSB ends in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

72. A method of detecting DSBs in a nucleic acid sample, the method comprising: exposing the nucleic acid sample to conditions suitable for in situ attachment of a first linker to DSB ends in the nucleic acid sample; exposing the nucleic acid sample to conditions suitable for attachment of a sequencing adapter that is suitable for single-molecule sequencing to the first linker in order to generate a nucleic acid library; and sequencing the nucleic acid library using single-molecule sequencing.

73. The method of claim 71 or claim 72, comprising alignment to a reference sequence of sequence reads generated by the sequencing; and the identification of the location of DSBs within the nucleic acid sample.

74. The method of any one of claims 1 to 73, wherein the method comprises detecting the presence / absence of epigenetic modifications.

75. A kit for preparing a nucleic acid library for detecting double-strand breaks (DSBs) in a nucleic acid sample, the kit comprising: a linker, and optionally a sequencing adapter suitable for single-molecule sequencing.

76. The kit of claim 75, wherein the kit comprises components for carrying out the method of any one of claims 1 to 74.

77. A nucleic acid linker molecule, wherein one end is suitable for ligation to a DSB end and the other end is suitable for ligation to a sequencing adapter.

78. The nucleic acid linker molecule of claim 77, wherein the nucleic acid linker molecule is suitable for use as the first linker in the method of any one of claims 1 to 74.

Images