Detection method for nucleic acid single-strand breakage

Abstract

The invention relates to a method for detecting a single-stranded break (SSB) site in double-stranded (ds) DNA. A dsDNA sample is contacted with an oligonucleotide probe comprising a first sequencing linker and a tag, and then contacted with a DNA ligase. And the DNA ligase is used for covalently linking the oligonucleotide probe to the dsDNA chain at the dsDNA single-chain breakage site. The dsDNA sample is then contacted with a transposase loaded with a second sequencing linker such that the transposase cleaves the dsDNA to produce a population of DNA fragments comprising the second sequencing linker. DNA strands comprising the first and second sequencing linkers are isolated from the population and the sequence of the isolated DNA strands is determined. These methods and kits for carrying out these methods are provided.

Description

[0001] field

[0002] This invention relates to a method for detecting and locating single-strand break (SSB) sites in double-stranded nucleic acids (such as double-stranded deoxyribonucleic acid (dsDNA)).

[0003] background

[0004] Shortly after the elucidation of DNA structure, the influence of endogenous gaps on DNA became readily apparent (Lindahl and Nyberg, 1972), resulting from various processes including oxidation, hydrolysis, alkylation, DNA base mismatches, and changes in mediating cellular responses. It is estimated that up to 70,000 gaps occur per cell per day (Lindahl and Barnes, 2000). Although these DNA gaps have historically been associated with mutations and various diseases, including cancer (Vilenchik et al., 2003), the role of endogenous DNA gaps in normal cellular function has recently gained increasing recognition (Wu et al., 2021).

[0005] Therefore, several techniques have been developed to measure the genomic distribution and frequency of DNA breaks. Most of these techniques are capable of measuring the genomic map of double-stranded DNA breaks with reasonable accuracy (Yan et al., 2017; Amente et al., 2021). Although single-stranded DNA breaks are ubiquitous (Lindahl and Barnes, 2000), few methods provide whole-genome detection of single-stranded DNA breaks. These methods include SSiNGLe (localizing single-strand breaks at the nucleotide genome level; Cao et al., 2019), SSB-Seq (Baranello et al., 2014; Baranello et al., 2018)), XR-Seq (Hu et al., 2015), and GLOE-Seq (Sriramachandran et al., 2020). These methods always rely on capturing the 3'OH end of the DNA break to locate its position, and since the 3'OH end is also present at the site of double-stranded DNA breaks, the DNA break measurement results may not represent single-stranded DNA breaks, but may instead reflect nicks in the DNA.

[0006] Simple techniques are still needed to quickly and accurately locate SSBs.

[0007] Overview

[0008] The inventors have developed a method (referred to herein as “DNA GAPS sequencing” or “GAPS-seq”) that allows for rapid, high-resolution detection of single-strand breaks (SSBs) in double-stranded (ds) DNA, and can be used, for example, to locate or assess SSBs in genomic DNA in a strand-specific manner.

[0009] A first aspect of the present invention provides a method for detecting single-strand break (SSB) sites in double-stranded (ds) DNA, comprising:

[0010] (I) Contact the dsDNA sample with an oligonucleotide probe containing a first sequencing adapter and a tag.

[0011] (ii) Contact the dsDNA sample with DNA ligase, so that the DNA ligase covalently links the oligonucleotide probe to the dsDNA strand at the single-strand break site of the dsDNA.

[0012] (iii) The dsDNA sample is contacted with a transposase loaded with a second sequencing adapter, causing the transposase to cleave the dsDNA to produce a population of DNA fragments containing the second sequencing adapter.

[0013] (iv) Isolate DNA strands containing the first and second sequencing adapters from the group.

[0014] as well as;

[0015] (v) Determine the sequence of the isolated DNA strands.

[0016] The sequence of the separated strands can indicate the location of SSBs within the dsDNA.

[0017] The first aspect of the method may include determining the sites of SSBs in first and second samples of dsDNA and / or determining the frequency of SSB occurrence. Differences in SSB sites or differences in SSB frequency at various sites may be determined between the first and second samples.

[0018] One of the first and second samples may have already received treatment, or it may be obtained from diseased cells or cells of a specific age or developmental stage. The other sample may serve as a reference or control. The effect of treatment, disease, developmental stage, or age on the location or frequency of SSBs can be determined.

[0019] A second aspect of the present invention provides a kit for detecting single-strand break (SSB) sites in double-stranded (ds) DNA; the kit comprises:

[0020] Oligonucleotide probes, which contain a first sequencing adapter and a tag,

[0021] DNA ligase,

[0022] transposases and

[0023] A second sequencing adapter loaded with or loadable onto a transposase.

[0024] The kit may further include binding components that specifically bind to the tag; sequencing primers, solid supports (such as beads), PCR reagents and / or other reagents.

[0025] Other aspects and embodiments of the invention are described in more detail below. Brief description of the attached diagram

[0027] Figure 1 This paper presents an example of a genome-wide DNA gap-specific measurement method (GAPS-Seq). Cell nuclei derived from cells or tissues are immobilized in situ and then ligated to biotinylated DNA adapters attached to hexamers (including their random sequences). The nuclei are then lysed, genomic DNA is fragmented and ligated to a second (R2) adapter, and the biotinylated fragments are captured with streptavidin and then separated into strands. PCR amplification, sequencing, and localization are then performed to reveal the genomic sites carrying the DNA gaps.

[0028] Figure 2 The results of gaps-seq measurements of ssDNA gaps are shown. GAPS-seq readings in HEK293 cells are shown as normalized to library size and then averaged in 40bp windows. The figure plots the average signal for all protein-coding genes relative to the transcription start site within a 40bp window. This line represents the mean of four replicates, and the shading represents the standard deviation.

[0029] Figure 3 This study presents an example of ssDNA gap distribution in etoposide-treated and untreated HEK293 cells. Etoposide treatment is known to increase endogenous DNA gaps by inhibiting topoisomerase. As predicted, GAPS-seq detected more ssDNA gaps in the etoposide-treated group compared to the control group treated with DMSO alone. Interestingly, GAPS-seq also detected more ssDNA gaps in enhancer sequences compared to surrounding regions, suggesting that the regulatory region undergoes a dynamic cycle of ssDNA gapping and repair.

[0030] Figure 4 TapeStation traces of DNA GAP-seq libraries obtained from different input samples are shown. Inputs range from 1 million to 100 cells.

[0031] Figure 5The sensitivity and specificity of GAPS-seq using restriction endonucleases are shown. The figure shows GAPS-seq reads in HEK293 cells treated with restriction endonucleases prior to GAPS-seq. Reads were normalized to library size and then averaged in 40 bp windows. The figure plots the average signal (relative to the midpoint of the site) of all restriction endonuclease sites in the human genome, in 40 bp windows. Each subplot shows a different restriction endonuclease site, and each colored line represents a different sample—each sample was treated with a different enzyme.

[0032] Figure 6 This demonstrates the specificity of GAPS-seq for single-strand gaps rather than DNA nicks. The figure shows GAPS-seq reads in BJF cells treated with nicking enzymes and exonuclease III prior to GAPS-seq. Reads were normalized to library size and then averaged in 40 bp windows. The figure plots the average signal (relative to the center of the site) for all nicking enzyme motif instances in the human genome, in 40 bp windows. The left subplot shows control cells treated with neither enzyme. The middle subplot shows cells treated with only nicking enzymes. The right subplot shows cells treated with both nicking enzymes and exonucleases.

[0033] Figure 7 The results of measuring DNA breaks at gene promoters at different expression levels using GAPS-seq are shown. GAPS-seq readings in HEK293 cells were normalized to library size and then averaged in a 400 bp window. This line represents the average of three replicates. This result demonstrates that GAPS-seq can detect the known relationship between DNA breaks and transcription.

[0034] Figure 8 The diagram shows a hierarchical clustering dendrogram obtained by analyzing different samples using GAPS-seq and quantifying at the gene promoter (+ / - 2kb of TSS). NPCs are neural progenitor cells derived from human embryonic stem cells differentiated in vitro. hESCs are human embryonic stem cells. HEKs are HEK293 cells. BJFs are the major human primary foreskin fibroblasts.

[0035] Figure 9 A scatter plot of the first two major components derived from GAPS-seq or sBLISS, quantified on gene promoters in HEK293 cells, is shown. The plot clearly illustrates the methods used to generate the library for sample separation, thus demonstrating that GAPS-seq measures different aspects of the genome compared to double-strand break analysis methods such as sBLISS.

[0036] Detailed description

[0037] This invention relates to a method for detecting single-stranded breaks (SSBs) in double-stranded (ds) nucleic acids (such as double-stranded DNA). A dsDNA sample is contacted with an oligonucleotide probe containing a first sequencing adapter and a tag, and then with a DNA ligase. The oligonucleotide probe is covalently attached to the dsDNA strand at a single-strand break or gap in the dsDNA by the DNA ligase. The dsDNA is then contacted with a transposase loaded with a second sequencing adapter. The transposase cleaves the dsDNA and produces a cluster of fragments with a second sequencing adapter at one or both ends. A DNA strand containing the second sequencing adapter at one end and the first sequencing adapter at the other end is then isolated from the cluster of fragments, and the sequence of the isolated DNA strand is determined (e.g., by sequencing). The sequence of the isolated strand may indicate the site of single-strand breaks (SSBs) within the dsDNA. Strand-specific SSB sites within the dsDNA in the sample can be identified or located from the sequence of the isolated strand.

[0038] The methods described herein can be used, for example, to distinguish SSBs from other genetic damage, to determine the location and / or frequency of SSBs in dsDNA (such as genomic DNA) samples, and to allow for high-resolution, strand-specific localization of SSBs.

[0039] The methods described herein can also be used to detect SSBs in low-input samples, from cells to tissue sections. Because cells or tissues do not require labeling prior to fixation, the methods described herein can directly detect newly formed gaps. Furthermore, sample fixation allows for sample storage and implementation of the methods described herein long after the initial sample collection without affecting the number of DNA SSBs or gaps detected. The sensitivity of the methods described herein allows for tracking the evolution of DNA SSBs or gaps with treatments (such as chemotherapy), developmental stages, and / or aging.

[0040] The methods described herein can be used with dsDNA in solution, cell-free dsDNA, or any cells or tissues that can be obtained in suspension, and allow for the rapid generation of sequencing libraries and the provision of DNA SSB or gap information, making these methods suitable for clinical applications. The methods described herein can also be used with dsDNA samples obtained, for example, from cells, organoids, or tissues, or, for example, cell-free DNA.

[0041] The method described herein is also compatible with bisulfite conversion and allows for the measurement of DNA methylation at sites near the nick. This allows for the assessment of the presence of the nick and DNA methylation, and the evaluation of the spatial relationship between the nick location and DNA methylation.

[0042] Single-strand nick (SSB) is a common type of DNA damage or gene defect. An SSB (interchangeably called a single-strand nick) is a discontinuous region (“broken strand”) in one strand of dsDNA, while the other strand (“intact strand”) remains undamaged. At an SSB, one or more phosphodiester bonds are missing in the broken strand, resulting in a discontinuity or break in the nucleotide chain that forms the remaining strand. At the SSB site, one or more nucleotides may be missing in the broken strand, resulting in a gap in the nucleotide sequence of the broken strand relative to the intact strand.

[0043] dsDNA can include any dsDNA, including, for example, genomic DNA, plasmid DNA, or viral DNA. Preferably, dsDNA is genomic DNA. For example, dsDNA can be a chromosome, a mini-chromosome, a whole chromosome, or a portion of multiple chromosomes, or derived from circulating DNA. Suitable dsDNA may also include mitochondrial DNA, plasmid DNA, or viral DNA inserted into a chromosome or genome.

[0044] Suitable dsDNA may include prokaryotic dsDNA, such as bacterial dsDNA. For example, the dsDNA used as described herein may include part or all of the genome of a prokaryotic cell (such as a bacterial cell), more preferably the entire genome of a prokaryotic cell.

[0045] Preferably, the dsDNA is eukaryotic dsDNA, such as mammalian dsDNA, such as human DNA. For example, the dsDNA used herein may comprise a portion or all of the nuclear and / or mitochondrial genome of a eukaryotic cell, more preferably the entire eukaryotic cell genome.

[0046] The dsDNA sample used as described herein may contain one or more, ten or more, one hundred or more, or one thousand or more cellular genomes. The dsDNA sample used as described herein may contain 3 pg, 6 pg or more, 60 pg or more, 600 pg or more, or 6 ng or more DNA.

[0047] In some embodiments, dsDNA samples can be obtained from tissues, organoids, cells, cell extracts, cellular components (e.g., organelles such as the nucleus or mitochondria), or biological fluids (e.g., blood, sperm, cerebrospinal fluid, amniotic fluid, pleural fluid, peritoneal fluid, or saliva). dsDNA can be cellular DNA or cell-free DNA (cfDNA).

[0048] In some implementations, dsDNA can be obtained from frozen cell or tissue samples.

[0049] In some embodiments, dsDNA can be within cells, cell nuclei, or nuclear extracts, such as isolated genomic DNA (gDNA). Cell nuclei or nuclear extracts can be obtained from cells using standard techniques. In some preferred embodiments, dsDNA can be within the cell nucleus. A sample containing one or more cell nuclei can be contacted with the oligonucleotide probe described herein, such that the probe contacts the dsDNA in the cell nucleus. This may allow for the localization of SSB sites within the cellular genome. For example, methods for localizing SSB sites within the cellular genome may include;

[0050] (i) Contact the cell nucleus with an oligonucleotide probe containing a first sequencing adapter and a tag.

[0051] (ii) Contact the cell nucleus with DNA ligase, allowing the DNA ligase to covalently link the oligonucleotide probe to the dsDNA strand at the single-strand break site of the dsDNA.

[0052] (iii) The cell nucleus is brought into contact with a transposase loaded with a second sequencing adapter, which then cleaves the dsDNA to produce a fragment cluster containing a terminal second sequencing adapter.

[0053] (iv) Isolate the DNA strand containing the first and second sequencing adapters from the group; and

[0054] (v) Determine the sequence of the isolated DNA strands.

[0055] The sequence of isolated DNA strands can indicate the location of SSBs within the cell genome.

[0056] In some implementations, the dsDNA used as described herein can be obtained from eukaryotic cells. Eukaryotic cells can be isolated, for example, as immortalized cell lines or primary cells obtained from an individual (such as a human subject), or can be in the form of tissues or organoids. For example, the nucleus can be obtained from eukaryotic cells in samples obtained from an individual (such as biopsy or xenograft samples).

[0057] Suitable eukaryotic cells can include mammalian cells, preferably human cells. For example, eukaryotic cells can include somatic cells and germline cells, and can be at any developmental stage, including fully or partially differentiated cells or undifferentiated or pluripotent cells, including stem cells such as adult or somatic stem cells, fetal stem cells, or embryonic stem cells. Suitable eukaryotic cells also include induced pluripotent stem cells (iPSCs), which can be derived from any type of somatic cell according to standard techniques. Eukaryotic cells can also include nerve cells (including neurons and glial cells); contractile muscle cells; smooth muscle cells; hepatocytes; hormone-synthesizing cells; sebaceous gland cells; pancreatic islet cells; adrenal cortical cells; fibroblasts; mesenchymal cells; epithelial cells; keratinocytes; endothelial cells; urothelial cells; osteocytes; chondrocytes; immune cells (e.g., leukocytes); mesothelial cells and adipocytes; bone marrow cells or samples thereof.

[0058] Suitable eukaryotic cells also include normal cells and disease cells, such as cells associated with disease conditions, including cancer cells (e.g., malignant tumors, sarcomas, lymphomas, blastomas, or germline tumor cells), and cells with genotypes of hereditary diseases (e.g., Huntington's disease, cystic fibrosis, sickle cell disease, phenylketonuria, Down syndrome, or Marfan syndrome).

[0059] Suitable eukaryotic cells also include embryonic cells, extraembryonic cells, and cell culture models.

[0060] In some implementations, the dsDNA used as described herein may be derived from diseased cells, such as cells obtained from an individual with a disease, like cancer cells, such as those obtained from a human subject. The frequency or distribution of SSBs within the dsDNA can indicate the diagnosis, severity, penetrance grade, or prognosis of a disease (e.g., cancer) in the individual from whom the dsDNA was obtained. Diseases or cancers may be caused or characterized by, for example, defects in DNA repair pathways.

[0061] In other embodiments, the dsDNA may be derived from cells at a specific developmental stage, such as cells obtained from an embryo; or from age-related cells, such as cells obtained from an individual of a specific age (e.g., an elderly person); or from treated cells. The treated cells may include cells treated with reprogramming factors (such as Oct3 / 4, Sox2, Klf4, and c-Myc (“Yamanaka factor” or “OSKM”), radiation, compounds, and drugs (e.g., chemotherapy drugs).

[0062] For example, the methods described herein can be used for cytotoxicity assays. Cytotoxicity assays may include stimulating cells and determining the frequency or distribution of gaps or SSBs in the cellular dsDNA. The frequency or distribution of gaps or SSBs in the cellular dsDNA may indicate the cytotoxicity of the stimulus. Suitable stimuli include exposure to compounds and radiation.

[0063] In other embodiments, dsDNA may be derived from cells treated with nucleases such as CRISPR nucleases (e.g., Cas9 or Cpf1). The frequency or distribution of SSBs within the dsDNA may indicate off-target effects of the nuclease in the treated cells.

[0064] In other embodiments, dsDNA can be derived from oocytes or sperm cells obtained from a donor (e.g., a human donor). The frequency or distribution of SSBs within the dsDNA can indicate the quality of the donor oocytes or sperm cells. This can be useful in, for example, assisted reproductive technology (ART) methods.

[0065] In some embodiments, cells may be fixed and permeated prior to step (i) of the method described herein.

[0066] Fixation can be useful, for example, to reduce background by mitigating SSB induced during sample processing before adding ligases. Suitable methods for fixing cells are well known in the art, including contacting cells with aldehyde fixatives (such as formaldehyde, formalin, or glutaraldehyde); or with alcohol fixatives (such as methanol, ethanol, or acetone). For example, cells can be fixed by exposure to 2% formaldehyde.

[0067] Permeation treatment can be useful, for example, to allow oligonucleotide probes and DNA ligases to contact dsDNA in cells, or to extract cell nuclei or dsDNA from cells. Suitable methods for permeation treatment of cells are well known in the art, including contacting cells with a detergent, such as 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., Triton X-100). TM ), nonylphenoxy polyethoxyethanol (such as NP-40) TM Polyoxyethylene sorbitan monolaurate (such as Tween™), saponins, or digitalis saponins. For example, cells can be permeated by exposing them to 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (e.g., 0.2% 2-[4-(2,4-trimethylpentan-2-yl)phenoxy]ethanol).

[0068] Prior to step (i), cell nuclei can be isolated or extracted from the fixed and permeabilized cells. For example, the method may include providing a cell sample and extracting cell nuclei from the cell sample. The cell nuclei or genomic DNA therein can then be used in the method of the present invention.

[0069] In some embodiments, the dsDNA sample may be in solution in the methods described herein. For example, dsDNA or cells containing nucleic acids may be contacted in solution with probes, DNA ligases, and / or transposases. The dsDNA, dsDNA-containing nucleic acids, or dsDNA-containing cells may be washed, for example, between steps by centrifugation and resuspension.

[0070] In other embodiments, in the methods described herein, dsDNA samples can be immobilized on a solid support. The solid support is an insoluble, non-gel-like material whose surface can immobilize capture molecules to capture dsDNA or cells containing dsDNA. Examples of suitable supports include glass slides, micropores, membranes, or microbeads. The support can be in particulate or solid form, including, for example, plates, test tubes, beads, balls, filters, fabrics, polymers, or membranes. The capture molecules may bind to proteins, glycoproteins, or other molecules on the cell surface. Capture molecules suitable for cells are well known in the art, including lectins that bind to extracellular glycoproteins on cells, such as concanavalin A.

[0071] The sequences of DNA strands isolated from the resulting fragment cluster can indicate the sequences of broken strands at SSB sites in the dsDNA. Multiple DNA strands can be isolated from the cluster. The sequences of the multiple isolated DNA strands can indicate the sequences of broken strands at multiple SSB sites in the dsDNA.

[0072] In the method described herein, the nucleic acid sample (e.g., dsDNA) is contacted with an oligonucleotide probe. The oligonucleotide probe includes a first sequencing adapter and a tag.

[0073] Sequencing adapters are double-stranded oligonucleotides that attach to nucleic acid molecules to allow sequencing, for example, by facilitating the amplification of nucleic acid molecules using sequencing primers. Suitable sequence adapters for any sequencing method are available in the art. In some embodiments, a sequencing adapter may include a primer recognition sequence. A primer recognition sequence is a nucleotide sequence complementary to an amplification primer sequence. The presence of a primer recognition sequence allows the adapter-tagged DNA strand to be amplified, for example, by using amplification primers targeting the primer recognition sequence. In dsDNA, the primer recognition sequence may be a non-naturally occurring heterologous sequence. Suitable primer recognition sequences are well known in the art, including IlluMina-compatible barcode i7 / i5 primers. A first sequencing adapter may include a first primer recognition sequence.

[0074] In some implementations, the first sequencing adapter may also include a barcode. A barcode is a nucleotide sequence that is unique to the sample from which the nucleic acid population is obtained. A suitable barcode sequence can be 6-10 nucleotides. Barcodes allow for the definitive identification of sequence reads from a specific sample in a pooled multiplex sequencing reaction. Each sample may have a unique barcode so that all nucleic acids from the same sample receive the same barcode. Once preparation is complete, nucleic acid populations from different samples can be pooled and sequenced. The sample from which the sequence reads generated from the original pool can then be identified from the barcode. For example, a suitable barcode for multiplex sequencing (24-fold reaction) of 24 samples may consist of at least 6 nucleotides, preferably 6 nucleotides (Craig DW et al., 2008, *Nature Methods* 5887; Cronn R et al., 2008, *Nucleic Acid Research*, Vol. 36, e122). The use of barcodes in sequencing reactions is well known in the art. For example, IlluMina sequencing 8-polymer barcodes i5 and i7 can be used. In other implementations, one or both sequencing primers may include barcodes.

[0075] A tag allows the separation of the DNA strand to which it is attached. Suitable tags include any marker, molecule, or group that allows the binding member to specifically bind to the generating fragment or generating strand to which it is attached. The tag may allow covalent binding of the binding member, or more preferably nonvalent binding. Suitable tags include immunogens, such as digoxin; short peptides, such as glutathione and FLAG. TM ; or small organic compounds, such as biotin and trimethoprim (TMP).

[0076] Preferably, the tag is biotin, and the oligonucleotide probe is a biotinylated oligonucleotide.

[0077] The oligonucleotide probe may further include a hybridization region. The hybridization region may contain 2-10 nucleotides, preferably 4-8 nucleotides, more preferably 6 nucleotides, which anneal to the intact strand of dsDNA at the SSB site.

[0078] In some implementations, the hybridization region can be nonspecific and can bind to the intact strand at any SSB site. For example, the hybridization region can have a random sequence. The nucleotides at each position in the hybridization region can be random (“N”; i.e., randomly selected from G, C, T, or A), resulting in diversity in the hybridization region of the oligonucleotide probe.

[0079] In other embodiments, the hybridization region can be specific and can bind to the intact strand at a particular SSB site. For example, in dsDNA, the hybridization region can be complementary to the intact strand sequence at a target site, for example, to assess or detect the site of the SSB. The target sequence can be the site of interest; or a known site of the SSB, such as a promoter.

[0080] Examples of oligonucleotide probes are shown in Table 4, which provides suitable ssBreak_3prime and ssBreak_5prime sequences.

[0081] Oligonucleotide probes are ligated to dsDNA strands containing gaps or SSBs (i.e., broken strands) using a DNA ligase. Suitable DNA ligases are well known in the art and are available from commercial suppliers. Examples of DNA ligases suitable for the methods described herein include T4 DNA ligase, T7 DNA ligase, ligase 3, and ligase 1. In one embodiment, the methods described herein are performed using T4 DNA ligase.

[0082] The dsDNA may be contacted simultaneously or more preferably sequentially with an oligonucleotide probe and a DNA ligase, wherein the dsDNA is contacted with the oligonucleotide probe and then with the DNA ligase.

[0083] The oligonucleotide probe can be contacted with dsDNA, causing the oligonucleotide probe to anneal to the intact strand of dsDNA at one or more nicks or SSB sites. Then, a DNA ligase can be contacted with the dsDNA, covalently attaching the oligonucleotide probe to the end of the broken strand at the nick or SSB site in the dsDNA.

[0084] In some implementations, the oligonucleotide probe is attached to the 3' end of the dsDNA break strand at a nick or SSB site. A suitable oligonucleotide probe for attachment to the 3' end may include a 5' phosphate group and a 3' tag. After transposase labeling, the second sequencing adapter may be located at the 5' end of the separated DNA strand, and the tag may be located at the 3' end of the separated DNA strand.

[0085] In other embodiments, the oligonucleotide probe is attached to the 5' end of the dsDNA break strand at a nick or SSB site. A suitable oligonucleotide probe for attaching the 5' end may contain a 5' tag and a 3' hydroxyl group. After transposase labeling, the second sequencing adapter may be located at the 3' end of the separated DNA strand, and the tag may be located at the 5' end of the separated DNA strand.

[0086] Oligonucleotide probes are ligated to the broken strand at the SSB site, and then dsDNA is fragmented using transposase to produce a cluster of dsDNA fragments with a terminal second sequencing adapter tag.

[0087] In some embodiments, the transposase can be an activatable transposase. An activatable transposase can be converted from an inactive state to an active state by changing conditions. For example, by increasing magnesium ions (Mg... 2+ or manganese ions (Mn) 2+ When the concentration of magnesium is increased, for example to a concentration in the range of about 0.1 mM to about 10 mM, the magnesium-activated transposase can be converted to its active state.

[0088] Suitable activatable transposases are well known in the art, including Tn5, Tn7, Mu, IS5, and IS91 (e.g., patent US9005935B2, Mizuuchi, K. published in *Cell*, Vol. 35, p. 785, 1983; Savilahti, H et al., *European Organization for Molecular Biology Journal* (EMBO J), Vol. 14, p. 4893, 1995; Goryshin and Reznikoff published in *Journal of Biochemistry* (J. Biol. Chem), Vol. 273, p. 7367 (1998); and patent WO2022056309A1). In some preferred embodiments, the transposase may be a Tn5 transposase. Tn5 transposases are available from commercial suppliers (e.g., Tagmentase). TM Company, Diagenode TM company).

[0089] Transposases can be loaded with one or more second sequencing adapters. The transposase can be preloaded with a second sequencing primer, or the method may include loading a second sequencing primer into the transposase. The second sequencing adapter can be non-covalently bound to the transposase to form a complex comprising the transposase and one or more second sequencing adapters. Suitable methods for loading second sequencing adapters into transposases are well known in the art. For example, the second sequencing adapter can be incubated with the transposase at room temperature for 1 hour.

[0090] The sequencing adapter is as described above. A suitable second sequencing adapter for attachment to a transposase can be a double-stranded oligonucleotide containing a transposase recognition sequence and a primer recognition sequence. The transposase recognition sequence is the sequence in which the transposase targets the transposition process. For example, an inverse transposase recognition sequence may use a transposon sequence inserted by the transposase as a scaffold. The sequence of the transposase recognition sequence may depend on the transposase. Transposase recognition sequences suitable for different transposases are well known in the art. For example, suitable transposase recognition sequences for Tn5 may include 19-mer end sequences, such as an outer end (OE) sequence (e.g., 5'-CTGACT CTT ATA CAC AAG T-3') and an inner end (IE) sequence (e.g., 5'-CTG TCT CTT GAT CAG ATC T-3'); and a chimeric end (ME) (e.g., 5'-CTG TCT CTT ATA CAC ATC T-3'). In some preferred embodiments, a 19-mer ME sequence may be used. The second sequencing adapter may include a transposase recognition site and a second primer recognition sequence. Suitable sequencing adapters are well known in the art (e.g., N7 Nextera). TM (Connector).

[0091] Table 4 shows examples of suitable second sequence connectors, which detail the N7_top sequence and N7_bottom sequence.

[0092] A transposase cleaves a dsDNA sample to produce a cluster of DNA fragments. The DNA fragments in this cluster are labeled with a second sequencing adapter at one or both ends. Alternatively, the DNA fragments in this cluster may be labeled with a second sequencing adapter at both ends, and these fragments may not contain nicks or SSB sites. A DNA strand from a cluster of fragments containing nicks or SSB sites is labeled with a second sequencing adapter at one end and with an oligonucleotide probe containing a first sequencing adapter at the other end.

[0093] DNA strands labeled with the first and second sequencing adapters can be separated from the population using tags. The fragments can be denatured using any suitable technique, such as NaOH treatment, to produce DNA strands. The DNA strands labeled with the first and second sequencing adapters can then be separated using binding members that bind to the tag of the oligonucleotide probe.

[0094] A binding member is a molecule that specifically binds to a target molecule or ligand (such as a tag). The binding member may be covalently bound to, or more preferably non-covalently bound to, the tag of an oligonucleotide probe. For example, when the tag is biotin, the binding member may specifically bind to biotin. Suitable binding members for binding biotin tags may include anti-biotin antibodies, anti-biotin proteins, and streptavidin. A binding member that specifically binds to a tag should not exhibit any significant binding to molecules other than the tag. In particular, the binding member should not exhibit significant binding to proteins, DNA, or other antigens that may be present in cells or cell extracts. Typically, the binding affinity (Ka) of the antibody or other binding member that specifically binds to the tag can be greater than about 10. 5 moles per liter (e.g., 10) 6 or more, 10 7 or more, 10 8 or more, 10 9 or more, 10 10 or more, 10 11 or more or 10 12 (or more moles / liter). Suitable bonding components are well known in the art and can be produced using standard techniques or obtained from commercial sources.

[0095] In a preferred embodiment, the label is biotin, and the binding member is streptavidin. In some embodiments, the binding member may be attached to a solid support, such as a bead.

[0096] At any step of the methods described herein, a washing step may be performed as needed to remove unbound reagents. For example, after the contact step, oligonucleotide probes that are not bound to dsDNA may be removed by washing. Similarly, DNA ligases and / or transposases may also be removed by washing. Suitable washing methods are well known in the art. For example, as described herein, washing may be performed using buffers such as 20 mM HEPES pH 7.5, 150 mM NaCl, and 0.5 mM spermidine.

[0097] The isolated DNA strands generated as described above can be amplified to produce amplified products for sequencing. The isolated DNA strands can be amplified using primers that hybridize to the primer recognition sequence of the sequencing adapter. Suitable amplification methods are well established in the art and include polymerase chain reaction (PCR) (e.g., in “PCR Experimental Protocols; Methods and Applications Guide,” edited by Innis et al., 1990, Academic Press, New York; Mullis et al., Cold Spring Harbor Symposium on Quantitative Biology, Vol. 51, p. 263, 1987; Ehrlich (ed.), PCR Techniques, Stockton Press, New York, 1989; and Ehrlich et al., Science, Vol. 252, pp. 1643-1650, (1991)).

[0098] Amplification can produce a cluster or library of amplified products for sequencing. In dsDNA, each amplified product in the cluster or library may contain the nucleic acid sequence at the SSB site. The cluster or library of amplified products generated as described herein can be purified using standard techniques prior to sequencing, including spin column chromatography (such as Ampure XP). TM bead).

[0099] The sequence of the isolated strand or amplification product can be determined by any suitable technique. Suitable techniques include sequencing and hybridization-based techniques, preferably sequence-specific amplification, such as qPCR.

[0100] In a preferred embodiment, the sequence of the isolated strand or amplification product can be determined by sequencing the strand or product.

[0101] Separated DNA strands or amplified products can be sequenced using standard sequencing technologies. Suitable technologies include any convenient low-throughput or high-throughput sequencing technology or platform, including Sanger sequencing, Solexa IlluMina sequencing, and ligation-based sequencing (SOLiD). TM ), pyrosequencing; single-molecule real-time sequencing (SMRT) TM PacBioscience sequencing; and semiconductor array sequencing (Ion Torrent) TM Preferably, sequencing is performed using next-generation sequencing technology. Suitable experimental protocols, reagents, and apparatus for nucleic acid sequencing are well known in the art and are commercially available.

[0102] The sequence of the isolated DNA strand or amplification product can indicate the nucleotide sequence of the SSB site in dsDNA. For example, the sequence of the isolated DNA strand or amplification product may include the nucleotide sequence of the strand in which the SSB site occurs. The location of the SSB in dsDNA can be identified or located from the sequence of the isolated DNA strand or amplification product. For example, the location of the SSB in the genome of a cell (e.g., eukaryotic, mammalian, or human cell) can be identified or located using the methods described herein. The sequence of the isolated strand or amplification product can indicate the frequency of an SSB occurrence at a single site in dsDNA, or the probability of an SSB occurring at a single site in dsDNA.

[0103] In some embodiments, the methods described herein may include locating SSB sites within a first dsDNA and a second dsDNA, and identifying SSB sites present in the first dsDNA but not in the second dsDNA, or present in the second dsDNA but not in the first dsDNA. The methods described herein may include locating the SSB frequencies of sites within the first and second dsDNA, and identifying sites where the frequency of SSBs increases in the first dsDNA relative to the second dsDNA, or in the second dsDNA relative to the first dsDNA.

[0104] The first dsDNA can be a test sample, and the second dsDNA can be a reference or control sample. In some implementations, the first dsDNA can be derived from diseased cells, such as cells obtained from an individual with a disease, such as cancer cells, and the second dsDNA can be derived from normal or healthy cells.

[0105] The frequency or distribution of SSBs within the first dsDNA relative to the second dsDNA can indicate the diagnosis, severity, penetrance grade, or prognosis of a disease (e.g., cancer) in an individual who has acquired the first dsDNA. The disease or cancer may be caused by or characterized by defects in DNA repair pathways.

[0106] In other embodiments, the first dsDNA may be derived from cells at a specific developmental stage, such as cells obtained from an embryo, and the second dsDNA may be derived from adult or mature cells. In other embodiments, the first dsDNA may be derived from age-related cells, such as cells obtained from an individual of a specific age (e.g., an elderly person), and the second dsDNA may be derived from a control individual. In other embodiments, the first dsDNA may be derived from treated cells, and the second dsDNA may be derived from untreated control or reference cells. For example, the first dsDNA may be derived from cells treated with reprogramming factors (e.g., Oct3 / 4, Sox2, Klf4, and c-Myc (the “Yamanaka factor” or “OSKM”), radiation, compounds, and drugs (e.g., chemotherapy drugs).

[0107] In some implementations, the methods described herein can be used to stratify cells based on their age and / or disease state, such as cells obtained from test subjects (e.g., human subjects).

[0108] In some embodiments, the methods described herein can be used in tests to detect the therapeutic effect of drugs used to treat infections, for example, where the subject (e.g., a human subject) is infected with a virus or bacteria. In such a test, viral or bacterial dsDNA is obtained from the subject treated with an antiviral or antibacterial drug, and the methods described herein are then applied to the dsDNA to detect SSBs. The test results can be compared to a control sample containing the same viral or bacterial DNA that has not been exposed to any antiviral / antibacterial drug. This test can be used to determine the effectiveness of anti-infective drugs (e.g., antiviral or antibacterial drugs, such as antibiotics).

[0109] The preparation of dsDNA can be performed using standard methods known in the art.

[0110] In some embodiments, preparing dsDNA from any type of cell described herein may include one or more of the following steps that facilitate the introduction of oligonucleotide probes:

[0111] (i) Washing and lysis steps can be performed on cell samples containing dsDNA, for example, in containers such as small-volume test tubes, vials or coverslips, for example about 0.1 ml to about 2.0 ml, for example about 0.2 ml, or about 0.5 ml or about 1.5 ml.

[0112] (ii) The sample containing dsDNA can then be subjected to a buffer replacement step (e.g., by centrifugation or dilution of the sample).

[0113] The above buffer replacement can be achieved using the following method:

[0114] First, the cells are suspended in PBS (or a similar isotonic buffer). To separate the nuclei from the cells, they are centrifuged, the PBS is removed, and the cells are resuspended in a hypotonic lysis (or nucleolysis) buffer containing a mild detergent that causes the cell membrane to dissolve but leaves the nuclear membrane intact. Next, the cells are centrifuged again and resuspended in a second lysis buffer containing a stronger detergent (SDS), which dissolves proteins and thus removes chromatin proteins from the DNA (this step is called nucleosome depletion). After this, the nuclei are centrifuged again and resuspended in an SDS-free buffer. This stage is repeated several times to wash the nuclei and ensure SDS removal (as the presence of SDS inhibits downstream reactions).

[0115] In some implementations, the preparation of dsDNA from cells may include the following steps to facilitate dsDNA purification;

[0116] According to the method detailed herein, after contacting the dsDNA sample with a DNA ligase, the sample can be purified, for example, by digestion with a protease, such as by digestion with proteinase K, or by incubating the sample with proteinase K for about 2 hours to about 30 minutes, such as about 30 minutes.

[0117] In some implementations, magnetic beads can be used to separate DNA strands containing first and second sequencing adapters, wherein a sample containing dsDNA is mixed with magnetic beads and incubated for about 1 hour to about 2 hours, for example about 30 minutes.

[0118] In some embodiments, the present invention provides a method for detecting SSB breaks in dsDNA obtained from cells (e.g., any cells detailed herein), comprising the following steps:

[0119] (i) Optionally fix the cells,

[0120] (ii) The cells are subjected to washing and lysis steps, for example in a test tube / vial / coverslip with a volume of about 0.1-0.3 ml, such as about 0.2 ml, and then...

[0121] (iii) Replace the buffer in the sample containing dsDNA, for example by centrifuging the sample or by dilution.

[0122] (iv) Contact the dsDNA sample with an oligonucleotide probe containing the first sequencing adapter and tag.

[0123] (v) Contact the dsDNA sample with DNA ligase, allowing the DNA ligase to covalently link the oligonucleotide probe to the dsDNA strand at the single-strand break site.

[0124] (vi) Purify dsDNA, for example by digestion with a protease, such as by using proteinase K, for example by incubating the sample with proteinase K for approximately 2 hours to approximately 30 minutes.

[0125] (vii) The dsDNA sample is brought into contact with a transposase loaded with a second sequencing adapter, causing the transposase to cleave the dsDNA to produce a fragment cluster containing the terminal second sequencing adapter.

[0126] (viii) Isolate the DNA strands containing the first and second sequencing adapters from the group, for example by incubation with magnetic beads, for example, for about 5 minutes to about 2 hours, or for example, about 30 minutes.

[0127] (ix) Determine the sequence of the isolated DNA strands.

[0128] In some of the embodiments described above, the oligonucleotide incorporation and ligation steps can be performed directly on purified DNA or an intact cell nucleus.

[0129] The number of cells in step (i) or (ii) above can be in the range of about 10,000 to about 1 million, for example about 25,000, about 50,000, about 75,000, about 100,000, about 150,000, or about 200,000.

[0130] Protease digestion can be carried out in a temperature range of about 37°C to about 60°C, for example, when using the heat-sensitive proteinase K, it is carried out at about 37°C.

[0131] Steps (i) to (ix) above allow for the use of fewer cells to implement methods for detecting SSBs, and also allow for the implementation of these methods in a shorter time.

[0132] High-throughput approach: The method for detecting SSBs described in this article is applicable to achieving high-throughput methods. To scale this method to higher sample numbers, cells can be collected and processed in 96- or 384-well PCR plates (or using microfluidic devices). Oligonucleotides can be redesigned to include barcode sequences specific to each sample (i.e., 96 different barcodes used on a 96w sample plate). After ligating the barcode oligonucleotides, all samples can be pooled together (e.g., rapidly transferred to a V-groove Clickbio CBVBLOK200 using a centrifuge) for purification and all other downstream processing. After sequencing and data processing, samples can be identified by their barcode sequences. The process can also be made more efficient using automated liquid processors. Buffer replacement can also be eliminated instead of adding new buffer and diluting the previous components, reducing the number of pipetting steps required.

[0133] Single-cell method: The methods for detecting SSB described herein can be performed on single cells, including, for example, when using the methods described above to optimize the introduction of oligonucleotide probes and the purification of dsDNA to obtain dsDNA, generally following detailed instructions:

[0134] Samples containing dissociated, fixed cells are flow-cytosorted to separate individual cells into single wells of a 96- or 384-well plate containing lysis buffer required for nuclear dissociation and nucleosome depletion. As with the high-throughput methods described above, subsequent steps are performed in a PCR plate, using barcoded oligonucleotides to collect samples. Since cell loss is observed during centrifugation / buffer replacement, buffer removal replacement is preferred over buffer addition. When buffer addition is used, cells are collected into lysis buffer after lysis incubation, followed by the addition of another buffer that dilutes or inactivates the lysis reagent. Following this step, oligonucleotides and ligation reagents can be added, ensuring that the salt and detergent concentrations are suitable for ligase activity.

[0135] The methods described herein can be used, for example, in cytotoxicity assays. Cytotoxicity assays may include: applying a stimulus to cells and determining the frequency or distribution of gaps or SSBs in the cellular dsDNA. An increase in the frequency or distribution of gaps or SSBs in the cellular dsDNA compared to dsDNA from unstimulated control cells indicates that the stimulus is cytotoxic. Suitable stimuli include exposure to compounds and radiation.

[0136] In other embodiments, the first dsDNA may be derived from cells treated with a nuclease such as a CRISPR nuclease (e.g., Cas9 or Cpf1), and the second dsDNA may be derived from untreated control cells. The frequency or distribution of SSB within the first dsDNA relative to the second dsDNA can indicate off-target effects of the nuclease in the treated cells.

[0137] In other embodiments, the first dsDNA may be derived from an egg or sperm cell obtained from a donor, and the second dsDNA may be derived from a control egg or sperm cell. The frequency or distribution of the SSB within the first dsDNA relative to the second dsDNA can indicate the quality of the donor egg or sperm cell. This can be used, for example, in assisted reproductive technology (ART) methods.

[0138] Sequencing can produce a set of sequence reads of amplified or isolated DNA strands. For example, it can produce 1,000 or more, 10,000 or more, 100,000 or more, 10,000,000 or more, 100,000,000 or more, or 1,000,000,000 or more. Sequence reads can be analyzed using conventional bioinformatics techniques. Suitable techniques are well known in the art. For example, repetitive reads, low-quality reads, and reads caused solely by sequencing adapters may be deleted.

[0139] In some implementations, the presence of SSB sites in the sequence reads of the group, the frequency of SSB occurrence at the sites, and / or the pattern of SSB sites within the dsDNA can be analyzed. Further analysis can be performed to determine if other SSB-related features, such as mutations, epigenetic modifications, or sequence motifs, are present in the sequence reads of the group.

[0140] The sequence reads in this group can be located at one or more locations in a reference genome. Suitable reference genomes exist in the art. For example, human nucleic acid sequence reads in this group can be located at locations in the human genome sequence. In some embodiments, the reference genome can be matched with the sex, ethnicity, and / or other characteristics of the individual from whom the sample was obtained.

[0141] The fragmented sequence reads in this group are located by aligning them with sequences from a reference genome (e.g., the human genome). The locations of sequence reads in the reference genome can be identified. Suitable software tools for locating clusters of sequence reads within a genome are readily available in the art.

[0142] The distribution of sequence reads within the genome or at a set of loci or sites within the genome (i.e., the number of reads located at each location or set of loci or sites within the genome) can be determined based on the position of the reads within that set. Optionally, the distribution of sequence reads can be mathematically transformed.

[0143] Sample SSB maps can be generated from the distribution or transformation distribution of nucleic acid fragment sequence reads. A sample SSB map can include a set of scores or values ​​indicating the number or density of nucleic acid fragment sequence reads located at various sites or locations within the genome (i.e., a whole-genome map) or a set of loci or sites within the genome. The number or density of fragment sequence reads located at a particular site or location in the genome may indicate the frequency of SSBs at that site or location. Therefore, a sample SSB map can reflect the distribution of SSBs in the genome or at target loci within the genome. Sample SSB maps can be represented in any convenient form, such as numerical or graphical representation.

[0144] In some embodiments, sample SSB profiling can be used to identify SSB sites associated with biological responses to drugs or other compounds (such as cytotoxic responses) and to determine or predict an individual's response to drug or other compound treatments. Sample SSB profiling can be used for treatment stratification, optimization of combination therapies, diagnosis of disease conditions, determination of poor cellular repair in subjects (such as human subjects), and screening for treatments that can stimulate SSB repair and / or induce cell regeneration and / or reprogramming, for example, through exposure to agents (chemical and / or biological) that can stimulate SSB repair; determining side effects of drug or other compound treatments; cytotoxicity assays; determining off-target effects of nucleases; assisted reproductive technology methods; or studying aging or development. In some embodiments, SSB profiling can be used to differentiate different cell types, for example for cell type identification, a method that can be used in cell analysis or screening to discover cell type-specific differences in the genomic distribution of DNA gaps and to examine the effects of transcriptional interference on DNA gap formation dynamics.

[0145] Marker (target) sites are locations in the genome where the presence or frequency of SSBs varies across different sources; that is, SSBs at marker sites are source-specific, such as originating from different individuals, tissues, cell types, developmental stages, or ages. A set of marker sites in a sample SSB map can provide source-specific identification.

[0146] In some implementations, suitable marker sites can be identified by determining the presence of SSBs at multiple candidate sites in a reference SSB map from a set of reference sources. A reference SSB map may include a set of scores or values ​​indicating the SSBs at that set of marker sites in genomic DNA from a known source (e.g., a specific known tissue or cell type). A reference SSB map can reflect the presence of SSB sites in genomic DNA from known sources. Suitable reference SSB maps can be obtained or generated using routine experiments with known tissue or cell types, or generated from publicly available data sources such as genomic information databases.

[0147] Within a group, if a reference source exhibits a higher or lower frequency of SSB occurrence at a candidate site than other sources in the group, that candidate site can be identified as an SSB marker site. For example, the frequency of SSB occurrence at the site may be higher or lower than the average frequency of SSB occurrence in other reference sources within the group. In some implementations, the frequency of SSB occurrence at a site in a reference source may be higher or lower than a predetermined threshold relative to the average frequency of SSB occurrence at the corresponding site in other reference sources within the group.

[0148] In other embodiments, suitable marker sites can be identified by providing a first set of SSB maps from control individuals (e.g., healthy individuals) and a second set of SSB maps from test individuals (e.g., individuals with a disease (e.g., a specific cancer), individuals known to respond to or not respond to a drug, or individuals of a specific age). The SSB frequencies of multiple candidate sites in the first and second set of sample SSB maps can be compared. If the SSB frequency of a candidate site is higher or lower in the first set of sample SSB maps than in the second set of sample SSB maps, then that candidate site can be identified as a marker site. For example, the average frequency of SSB occurrence in the first set may be higher or lower than in the second set. In some embodiments, the average frequency of SSB occurrence at sites in the first set may be higher or lower than a predetermined threshold relative to the average frequency of SSB occurrence at sites in the second set.

[0149] If a candidate site is found to be in linkage disequilibrium with another site, and the site occurs more or less frequently at the reference source SSB than at sites from other sources in the group, then the candidate site can also be identified as a marker site.

[0150] SSB profiles from individual samples can be used to determine the presence or frequency of SSBs at a set of marker sites within an individual. This may help determine the efficacy of a drug or other compound on an individual or the individual's responsiveness to a drug or other complex, for example, to determine the therapeutic efficacy of a drug or other compound in an individual, or to determine the suitability of an individual for treatment with a drug or other complex. SSB profiles from individual samples can also be used to demonstrate the mechanism of action of a drug or other compound; as part of a clinical trial; or as evidence of off-target toxicity, where toxicity is caused by SSBs at other / alternate sites within dsDNA.

[0151] The methods described herein can be combined with other analytical techniques, such as in multi-genomic analyses. In some embodiments, the methods described herein may further include determining the presence of epigenetic markers at or near SSB sites in the dsDNA. Suitable epigenetic markers include modified nucleic acid bases, such as methylated DNA cytosine. For example, the isolated DNA strands produced as described herein can be treated with bisulfite or in combination with other chemical modifications (e.g., converting existing modifications to 5caC to distinguish 5hmC / 5mC) and sequenced. The sequences of the isolated DNA strands can be compared with a reference genome sequence, and modified cytosine residues in the isolated DNA strands can be identified.

[0152] The method described herein can be used instead of dsDNA with dsRNA or a DNA / RNA hybrid, wherein one strand is DNA and the other is RNA. When using dsRNA in the method described herein, the method can be modified as follows: an RNA ligase (e.g., T4 RNA ligase) can be optionally used instead of a DNA ligase, and RNA oligonucleotides can be optionally used instead of DNA oligonucleotides. After performing the ligation step, reverse transcriptase can be used to transcribe the RNA into DNA.

[0153] The dsRNA can be, for example, siRNA, mRNA, or tRNA. The methods described herein can be used, for example, as part of quality control, to assess the stability of dsRNAs (such as therapeutic siRNAs), including the stability of such RNA molecules when stored under different conditions, such as when stored in different formulations under different storage conditions, such as at different temperatures and / or for different times. When applied to dsRNA, these methods can also be used to identify RNA base modifications on modified RNA strands.

[0154] Screening methods: The methods for detecting SSBs described in this article can be used as screening methods, for example, to identify reagents (such as chemical and / or biological reagents, such as small molecule and / or peptide and / or protein therapeutics) that can repair SSBs in dsDNA and / or reprogram cells.

[0155] Therefore, a method for screening such reagents, such as small molecule and / or peptide and / or protein therapeutic agents, which can repair SSBs and / or reprogrammed cells in dsDNA, is also provided, the method comprising the following steps:

[0156] (a) Detect single-strand break (SSB) sites in double-stranded (ds) DNA control samples that have not been exposed to reagents (such as chemical and / or biological reagents, such as small molecules and / or peptides and / or protein therapeutics), and detect single-strand break (SSB) sites in test versions of the same dsDNA samples that have been exposed to reagents.

[0157] (i) Contact the test and control dsDNA samples with an oligonucleotide probe containing a first sequencing adapter and a tag.

[0158] (ii) Contact the test and control dsDNA samples with DNA ligase, allowing the DNA ligase to covalently link the oligonucleotide probe to the dsDNA strand at the single-strand break site of the dsDNA.

[0159] (iii) Contact the test and control dsDNA samples with a transposase loaded with a second sequencing adapter.

[0160] This allows transposases to cleave dsDNA to produce a fragment cluster containing a terminal second sequencing adapter.

[0161] (b) Isolate DNA strands containing the first and second sequencing adapters from the population in the test and control samples;

[0162] (c) Determine the sequences of isolated DNA strands from test and control samples.

[0163] (d) Compare the distribution of SSB spectra in the test sample and the control sample.

[0164] (e) Compared with the control sample, the separation and test sample showed a reduction in SSB-related chemical and / or biological reagents, and

[0165] (f) The isolated chemical and / or biological reagents are formulated into a pharmaceutical composition, for example, by adding appropriate pharmaceutically acceptable excipients.

[0166] The above-described pharmaceutical composition may be used to treat subjects (such as human subjects) to promote the repair of SSBs in cells and / or to reprogram the cells in the subjects.

[0167] The above-described SSB screening method can be applied to cells in test subjects, such as human cells, and may further include one or more of the following steps, such as facilitating the introduction of oligonucleotide probes:

[0168] (i) Cell samples containing dsDNA can be washed and lysed, for example, in test tubes or vials or coverslips with small volumes (e.g., about 0.1–0.3 ml, or about 0.2 ml).

[0169] (ii) The sample containing dsDNA can then be buffer-replaced, for example by centrifugation or dilution.

[0170] In some embodiments, the screening method described above may include the following steps to facilitate dsDNA purification: after contacting the dsDNA sample with a DNA ligase, the sample may be purified, for example by digestion with a protease, such as proteinase K, for example by incubating the sample with proteinase K for about 2 hours to about 30 minutes.

[0171] In some implementations, the separation of DNA strands containing the first and second sequencing adapters can be performed using magnetic beads. For example, a sample containing dsDNA can be incubated with magnetic beads for an incubation period of about 5 minutes to about 2 hours (e.g., about 30 minutes).

[0172] Protease digestion can be carried out in a temperature range of about 37°C to about 60°C, for example, when using the heat-sensitive proteinase K, it is carried out at about 37°C.

[0173] The screening methods described above can be used to identify reagents / compounds that can reprogram cells and / or repair SSBs present in dsDNA within cells. These screening methods can also be used to track the time-series process of cell reprogramming and / or repair, for example, by applying these methods to dsDNA obtained at different time points after incubation of the reagent / compound with dsDNA. The presence of nicks can be confirmed by replacing the reagent capable of repairing SSBs with a nick-forming reagent and then testing the dsDNA exposed to such a reagent against a control sample not exposed to the nick-forming reagent. Furthermore, the screening methods described above are also suitable for preparing and isolating dsDNA with designed-introduced nicks.

[0174] Kits for using the methods described herein and their use in such methods are also provided. Kits may include;

[0175] Oligonucleotide probes, which contain a first sequencing adapter and a tag,

[0176] DNA ligase,

[0177] transposases and

[0178] A second sequencing adapter loaded with or loadable onto a transposase.

[0179] The kit further includes binding components that specifically bind to the tag; sequencing primers and / or other reagents. The kit further includes suitable buffers and washing solutions; fixatives and permeabilizers.

[0180] The kit may further include a solid support. Suitable solid supports may include capture molecules that bind to eukaryotic cells, such as lectins; and / or binding components.

[0181] The kit may further include nucleic acid extraction and purification reagents. Suitable reagents are well known in the art, including rotary chromatographic columns.

[0182] The kit may further include amplification reagents. Suitable reagents are well known in the art, including primers, dNTPs, and thermostable polymerases. In some embodiments, the kit may include amplification primers for amplifying one or more target regions or sites in the genome.

[0183] The kit may include instructions for using the methods described herein.

[0184] Other aspects and embodiments of the invention provide for the foregoing aspects and implementations, wherein the term “comprising” is replaced by the term “consisting of…”, and the term “comprising” is replaced by the term “substantially consisting of…” in the foregoing aspects and implementations.

[0185] It should be understood that, unless the context otherwise requires, this application discloses all combinations of any of the foregoing aspects and embodiments. Similarly, unless the context otherwise requires, this application discloses single combinations of preferred and / or optional features, or combinations thereof, with other aspects.

[0186] After reading this disclosure, those skilled in the art will understand the modifications, further embodiments, and modifications thereof of the above embodiments, and therefore, all of the above are within the scope of this invention.

[0187] All files and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

[0188] As used herein, “and / or” should be considered a specific disclosure of each of two particular features or components, whether or not the other is included. For example, as listed separately herein, “A and / or B” should be considered a specific disclosure of (i) A, (ii) B, and (iii) A and B.

[0189] experimental

[0190] Example 1:

[0191] Materials and Methods

[0192] An example of the GAPS-Seq method is illustrated in, for example... Figure 1These examples include 1) cross-linking cells / tissues with cross-linking agents to immobilize DNA with single-stranded DNA gaps or SSBs; 2) using detergents to penetrate cells and denature chromatin proteins, thereby allowing access to genomic DNA containing SSBs; 3) directly ligating DNA oligonucleotide probes containing random hybridization regions, first sequencing adapters, and tags to sites in DNA gaps; 4) lysing cell nuclei to isolate genomic DNA; 5) adding second adapters to the isolated DNA using tagging techniques; 6) capturing the tagged adapters that bind to the DNA gap; and 7) isolating, amplifying, and sequencing the DNA strand containing the gap. Cells were briefly dissociated into single cells using TrypLE's Express Enzyme and then fixed in 2% formaldehyde at room temperature for 10 minutes. Cell nuclei were isolated on ice for 15 minutes using 0.2% Triton-X100 and then denatured chromatin proteins / DNA-binding proteins using SDS. The cell nuclei were then precipitated by centrifugation and resuspended in biotinylated GAPS-seq oligonucleotides containing the IlluMina Read 1 sequence. Then, T4 DNA ligase was added, and the suspension was incubated overnight at 16°C with shaking (at 800 rpm). Cross-linking and protein digestion were then reversed using proteinase K, followed by DNA purification on a column. Approximately 40 ng of the purified DNA was labeled with IlluMina Read 2, and the GAPS-seq fragment was captured on streptavidin beads prior to indexing PCR.

[0193] Sample preparation

[0194] 1M cells were isolated into single cells and fixed with 2% formaldehyde for 10 minutes at room temperature. The reaction was quenched by adding 2M glycine to a final concentration of 125mM, and the supernatant was removed by centrifugation at 100g for 5 minutes at 4°C. The cells were then resuspended twice in 1 mL phosphate-buffered saline (PBS), and the supernatant was washed by centrifugation at 100g for 5 minutes at 4°C. The cells were then resuspended in 1 mL of ice-cold LB1 (10mM Tris pH 8.0, 10mM NaCl, 1mM EDTA, 0.2% Triton) and incubated on ice for 15 minutes. The cells were then centrifuged at 300g for 5 minutes at room temperature to remove the supernatant. The nuclei were then resuspended in 1 ml of LB2 (10 mM Tris pH 8.0, 150 mM NaCl, 0.3% SDS) preheated to 37°C, and incubated in a 37°C hot mixer for 60 min. The mixture was then gently shaken at 400 rpm and centrifuged at 300 g for 5 min at room temperature to remove the supernatant. Fresh CTSX buffer (100 μL 10x Cutsmart, 10 μL 10% Triton X-100, 890 μL nuclease-free water) was prepared and heated to 37°C for resuspending the nuclei. The nuclei were examined by trypan blue staining and microscopic observation to ensure 100% trypan blue positivity and intact, rounded shape. The nuclei were then rotated at 300 g for 5 min at room temperature to remove the supernatant, and the CTSX washing was repeated.

[0195] Preparation of samples treated with etoposide

[0196] Etoposide treatment was performed to validate the method, as etoposide is known to induce gaps in both single-stranded and double-stranded DNA (Muslimovic et al., 2009). For this experiment, passage 14 HEK293 cells were treated with 30 μM etoposide added to the cell culture medium and incubated at 37°C for 4 hours in a 5% CO2 incubator. Control samples were treated with the same concentration of DMSO as the etoposide-treated cells and incubated at 37°C in a 5% CO2 incubator for 4 hours. Isolation and fixation were performed as described above.

[0197] Restriction endonuclease experimental sample preparation

[0198] Fix 1M cells (6 pg × 1M = 6 μg dsDNA) and process them exactly as described above. After washing in CTSX buffer, resuspend the cell nuclei in 100 μL CTSX buffer and add 2 μL of an appropriate restriction endonuclease. Incubate the samples overnight at the recommended temperature (37°C for all enzymes except TspRI, 65°C for TspRI), shaking at 400 rpm. Simultaneously process control samples that have not received any restriction endonucleases, and incubate the control samples at 37°C, shaking at 400 rpm.

[0199] Oligonucleotide ligation in GAPS-seq

[0200] Add 4 μL of 100 μM ssBreak oligo and 96 μL of the ligation mixture (formulation shown in Table 1 below) to ice, and then resuspend the cell nuclei.

[0201]

[0202]

[0203] Table 1

[0204] The cell nucleus and T4 ligase were incubated overnight on a shaker at 16°C and 400 rpm.

[0205] Reverse cross-linking agent and DNA purification

[0206] 10 μL of proteinase K (NEB) was added to the ligation reaction and incubated at 55 °C for 2 hours. Then, the DNA from the cell nucleus was purified using the zymoQuick DNA Microprep kit. 500 μL of DNA lysis buffer was added, and the nuclei were vortexed at 10000 g for 1 minute. The supernatant was transferred to a column and vortexed at 10000 g for 1 minute. 200 μL of pre-wash buffer was added to the column, and the column was centrifuged at 10000 g for 1 minute. Then, 500 μL of gDNA wash buffer was added to the column, and the column was centrifuged at 10000 g for 1 minute.

[0207] Then, 20 μL of water was added to the column, which was then placed in a 1.5 ml Eppendorf tube and centrifuged at maximum speed for 5 minutes at room temperature. DNA was then quantified using qubits, and the results were recorded. The labeling reaction was established as shown in Table 2 below.

[0208] TD buffer (IlluMina) 21μL N7 Tn5 12.5μM 1μL DNA 20μL

[0209] Table 2

[0210] The reactants were incubated at 55°C for 10 minutes, and then 10.5 μL of 0.2% SDS was added.

[0211] Biotin pull-down test on SSB samples

[0212] Prepare streptavidin beads. Wash 5 μL of each sample C1 bead twice in 2x B&W (10 mM Tris, 1 mM EDTA, 2 M NaCl) and resuspend in 50 μL of 2x B&W. Add 50 μL of beads to 50 μL of sample and mix on a shaker at room temperature for 1 hour. Remove the supernatant using a magnet and the beads.

[0213] Wash twice in 0.1M NaOH and twice in water. Resuspend the beads in the PCR mixture as shown in Table 3 below:

[0214] KAPA HiFi 2x readymix 10μL Bst3.0 (NEB) 1μL 10μMi7 index primer 1μL 10μMi5 index primer 1μL water 7μL

[0215] Table 3

[0216] The DNA on the beads was amplified according to the following steps: 65°C for 5 minutes; 98°C for 45 seconds, then 20 cycles; 98°C for 15 seconds, 60°C for 30 seconds, and finally 72°C for 1 minute. The PCR product was then purified with 0.65x AMPure XP beads (13 μL) and quality controlled (QC'd) on tapestation D5000 high-sensitivity tape.

[0217] Cell count dilution experiment

[0218] To meet the titration requirements, a series of diluted samples were prepared containing 1 million, 500,000, 100,000, 10,000, 1,000, 500, and 100 cells. All steps remained unchanged except for increasing the number of PCR cycles by 2 (100,000 cells), 3 (10,000 cells), 5 (1,000 cells), or 8 (500 and 100 cells).

[0219] Sequencing and data processing

[0220] Following the manufacturer's instructions, the library was assembled in equimolar amounts and then sequenced using an IlluMina NextSeq2000 instrument and P1 flow cytometry, with end reads of 50 nt and i7 and i5 reads of 8 nt. The sequencing reads were demultiplexed using the bcl2fastq2 (IlluMina) software to generate two FastQ files corresponding to read 1 and read 2. The FastQ files were processed using a Nextflow chip sequencing pipeline (Ewels et al., 2020) to generate BAM files containing the aligned reads. In short, this requires raw read QC using FastQC (v0.11.9) (Andrews 2010), followed by adjustments using Trim-Galore (v0.6.7) (Krueger et al. 2023) and cutadet (v3.4) (Martin et al. 2011) to remove the IlluMina primer sequence at the 3' end and the first 6 nt at the 5' end, which correspond to random primer fragments. The adjusted reads are then aligned with the GRCh38 version of the human genome using BWA (v0.7.17-r1188) (Li et al. 2010), and filtered using Samtools (v1.15.1) (Danecek et al. 2021) and picard (v2.27.4) to remove duplicates and reads located in blacklisted regions.

[0221] To analyze the promoter region, the BAM file was loaded into Seqmonk v1.48.1, and probe trend plots were generated using default parameters. The data was exported as a text file and imported into R (v4.1.2) (R Core Team 2022) for plotting using ggplot2 (v3.4.2) (Wickham 2009). To analyze restriction endonuclease sites, we first used the BSgenome.Hsapiens.UCSC.hg38 (v1.4.4) package in R and the matchPattern function from biostrings (v2.64.1) to locate all instances of a given restriction endonuclease motif in the GRC h38 genome. This was then input as an annotation track into the Seqmonk software, and probe trend plots were generated using the aforementioned promoters.

[0222] result

[0223] The GAP-seq method is used to determine the relative frequency of single-strand gaps at gene promoters. Figure 2 These data correspond to existing knowledge about the distribution of breaks in the genome and confirm that the GAP-seq method can successfully detect SSBs.

[0224] ssDNA nicks were analyzed in etoposide-treated and untreated HEK293 cells. Etoposide treatment is known to increase endogenous DNA nicks by inhibiting topoisomerase. The study found that GAPS-seq detected more ssDNA nicks in etoposide-treated samples compared to the control group treated with DMSO alone. Figure 3 Interestingly, compared to the surrounding region, GAPS-seq also showed more ssDNA gaps in the enhancer sequence, indicating that the regulatory region underwent a dynamic cycle of ssDNA gapping and repair.

[0225] Research has found that DNA GAP-seq can detect ssDNA gaps in libraries obtained from different input samples ranging from one million to 100 cells. Figure 4 ).

[0226] HEK293 cells were treated with restriction endonucleases to cut dsDNA at different motifs with preserved 3' or 5' overhangs. Using GAP-seq, the 3' end of the broken strand was targeted, successfully locating the cleavage site with the 5' overhang (BstEII HF; EcoR1 HF), but the blunt end cleavage site (SrfI) or the 3' overhang cleavage site (Apa1) could not be located. Figure 5 These figures show the average signal at all known restriction sites in the human genome.

[0227] The study found that the GAPS-Seq method described in this paper is a highly versatile and quantitative method for detecting DNA gaps across the entire genome at base resolution.

[0228] Example 2: Optimized Experimental Scheme - GAPS-seqv2

[0229] Further optimizations to the above protocol now allow the GAPS-seq method to complete in a shorter timeframe (24 hours instead of 48 hours) and with fewer steps. This updated experimental protocol (GAPS-seq v2) also additionally allows for a reduction in input cell count (e.g., approximately 100,000 instead of approximately one million).

[0230] The following describes the improvements from GAPS-seq v1 (in the example above) to v2:

[0231] After fixation, the cells were transferred to 0.2 ml PCR tubes for subsequent washing and lysis steps. Using smaller tubes reduces cell loss due to nuclei "adhering" to the tube wall.

[0232] Gap-seqv2 also allows:

[0233] Reduce all centrifugation times from 5 minutes to 2 minutes.

[0234] Simplified gDNA purification after overnight ligation. Proteinase K digestion time was reduced from 2 hours to 30 minutes, and the use of magnetic beads instead of columns allowed for the processing of more samples with less input material.

[0235] Specifically, the experimental protocol shall be implemented according to the following steps:

[0236] (i) After overnight ligation, the cell nuclei were precipitated by centrifugation at 500g for 2 minutes.

[0237] (ii) Remove the supernatant by pipetting, leaving about 10 μL of liquid in the test tube.

[0238] (iii) Add 1 μL of 8 U / μL proteinase K (NEB P8107S).

[0239] (iv) The samples were incubated at 55°C for 15 minutes and then at 80°C for 15 minutes.

[0240] (v) Add 35 μL of RLT Plus buffer (Kaijie, 1053393).

[0241] (vi) Add 45 μL of SPRI screening beads (Beckman, B23318), vortex thoroughly and incubate at room temperature for 10 minutes.

[0242] (vii) Place the sample on a magnet, and once the beads settle, remove the supernatant.

[0243] (viii) Wash the beads twice with 200 μL of 80% ethanol, and place the test tube on a magnet.

[0244] (ix) To elute gDNA, 25 μL of EB buffer (Kaiger, 19086) was added to the beads on the magnet and vortexed to mix.

[0245] (x) Place the test tube on a thermostat mixer and treat at 50°C and 2000 rpm for 10 minutes.

[0246] (xi) Then place the test tube on the magnet, and once the beads settle, transfer the liquid to a clean PCR test tube.

[0247] (xii) Optimized labeling reaction: Tn5 is now loaded using 4 μL of 12.5 μM N7 (purchased from Diagenode catalog C01070010-10) instead of 1 μL. This results in greater library complexity.

[0248] (xiii) The sample was conjugated with streptavidin beads for about 15 minutes, not 1 hour.

[0249] The optimized experimental protocol described above has been performed on multiple cell lines, examples of which are described in detail below. The number of cells used ranges from 10,000 to 1,000,000 cells.

[0250] Example 3: Experiments with nicking enzymes and exonucleases

[0251] GAPS-seq aims to analyze single-strand gaps in gDNA, rather than nicks, because nicks are more numerous and have less physiological impact. To determine whether GAPS-seq is sensitive to nicks or gaps, the following experiment was designed and performed. BJ cells [cell type: human foreskin fibroblasts; source: ATCC; catalog number: CRL-2522; batch number: 70027151] were prepared according to the GAPS-seq v2 cell fixation and lysis assay protocol described in Example 2 above. The cell nuclei were then treated with 0.1 μL / μL of the nick endonuclease Nt.BbvCI (NEB R0632S) in 1x rCutSmart buffer (NEB, B6004) at 37°C for 1 hour. Next, the cell nuclei were precipitated by centrifugation (as described above, at 300g for 2 minutes), washed twice in CTSX buffer, and then resuspended in 1x rCutSmart buffer (NEB, B6004) containing 1 U / μL exonuclease III (NEB, MO206S) and incubated at 37°C for 1 hour. The nuclei were washed twice more in CTSX buffer, and the 5-primer GAPS-seq v2 experimental protocol was continued starting with the addition of the oligomer and ligation master mix. The control group consisted of (a) cell nuclei treated with only the nicking enzyme and no exonuclease, and (b) cell nuclei not treated with either enzyme. Plotting was performed at the Nt.BbvCI recognition site (CCTCAGC), as described in the restriction enzyme experiments above. The combination of these two enzymes produced a single-stranded nick at the Nt.BbvCI recognition site, whereas treatment with the nicking enzyme only produced a nick in the DNA, which was intentionally ignored by GAPS-seq. Therefore, this experiment demonstrates that GAPS-seq is specific for single-stranded DNA gaps and does not analyze gaps in DNA. Figure 6 ).

[0252] Example 4: Transcription initiation site map stratified by gene expression level

[0253] It is known that both double-stranded and single-stranded gaps are enriched at transcription start sites in a manner dependent on the corresponding gene expression level. To confirm that GAPS-seq can detect this phenomenon, GAPS-seq-v2 was performed on HEK293 cells as described above. For plotting, the GAPS-seq BAM file (i.e., the localized sequencing reads) was read into R (https: / / bioconductor.org / packages / release / bioc / html / Rsubread.html) using the Rsubread package and overlapped with a 10kb region around the transcription start sites of all genes. The genes were then stratified into three groups based on their expression levels derived from publicly available HEK293 cell RNA-seq (https: / / doi.org / 10.1093 / nar / gky861): 1) highly expressed genes (500 or more raw counts); 2) moderately expressed genes (raw counts between 10 and 500); and 3) poorly expressed genes (raw counts less than 10). Then, the GAPS-seq signals were averaged at a window exceeding 400 bp, and then the averages were calculated for all genes (for each group separately) and plotted as a line graph. Figure 7 The study found that, since transcriptional levels are correlated with basal DNA breakage levels, this suggests that this method could be used, for example, to examine the effects of transcriptional perturbations on DNA gap formation dynamics, and vice versa.

[0254] Example 5: GAPS-seq can distinguish cell types

[0255] GAPS-seq has the potential to reveal cell type-specific DNA gaps and to be used for cell type differentiation. To demonstrate this, GAPS-seq was performed on (1) HEK293 cells, (2) BJ fibroblasts, and (3) human embryonic stem cells (ESCs) and NPCs derived from ESC cultures. Library preparation, sequencing, and data processing were as described in Gap-seq version 2 above. To construct a dendrogram, GAPS-seq signals were quantified in promoter regions (+ / - 2kb from the TSS of all protein-coding genes), hierarchical clustering was performed using the dist and hcl ust functions in R, and plotting was done using the ggdendro package [available at https: / / cran.r-project.org / web / packages / ggdendro / index.html]. The hierarchical clustering clearly separated the samples by cell line origin, thus demonstrating the practicality of GAPS-seq in cell type identification. Figure 8The results show that GAPS-seq can clearly distinguish different cell types, which may help in cell type identification and suggests that the method can be used to discover cell type-specific differences in the genomic distribution of DNA gaps.

[0256] Example 6: The GAPS-seq spectrum and the double strand breakage spectrum analysis method sBLISS are different.

[0257] Methods for analyzing double-strand breaks exist, such as sBLISS. To test the unique genomic signature of double-strand breaks in GAPS-seq, sBLISS was performed on the same HEK293 cell batch as GAPS-sex. The sBLISS experimental protocol followed that described by Bouwman et al. in Nature Protocols, Vol. 15, pp. 3894–3941 (2020). To compare the signals produced by the two methods, we overlapped the signals with promoter regions (from TSS to + / - 2kb) and then performed principal component analysis using the prcomp function in R (v4.1.2) (R Core Team 2022) https: / / cran.r-project.org / . The results are as follows: Figure 9 As shown in the figure, the separation of samples by the methods used to generate the library is clearly illustrated, thus demonstrating that the GAPS-seq method is measuring aspects of the genome differently than double-strand break analysis methods (such as sBLISS).

[0258] References

[0259] Lindahl T, Nyberg B. Purification of natural deoxyribonucleic acid, *Journal of Biochemistry*, September 12, 1972; Vol. 11 (No. 19): 3610-3618. doi: 10.1021 / bi00769a018. PMID: 4626532

[0260] Lindahl T, Barnes DE. Repairing Endogenous DNA Damage. Proceedings of the Cold Spring Harbor Quantitative Biology Symposium, 2000; Vol. 65: 127-133. doi: 10.1101 / sqb.2000.65.127.PMID: 12760027

[0261] Amente S, Scala G, Majello B, Azmoun S, Tempest HG, Premi S, Cooke MS. Genome-wide localization of genomic DNA damage: methods and implications. Cellular & Molecular Life Sciences, November 2021; Vol. 78 (Nos. 21-22): 6745-6762. doi: 10.1007 / 00018-021-03923-6. Electronic publication date: August 31, 2021. PMID: 34463773; PMCID: PMC8558167.

[0262] Yan W, Mirzazadeh R, Garnerone S. et al., “BLISS: A Multifunctional Quantitative Method for Genome-Wide Analysis of DNA Double-Strand Breaks,” *Nature Communications*, Vol. 8, p. 15058 (2017). https: / / doi.org / 10.1038 / ncomms15058

[0263] Wu W, Hill SE, Nathan WJ, Paiano J, Callen E, Wang D, Shinoda K, van Wietmarschen N, Coló N-Mercado JM, Zong D, De Pace R, Shih HY, Coon S, Parsadanian M, Pavani R, Hanzlikova H, Park S, Jung SK, McHμgh PJ, Canela A, Chen C, Casellas R, Caldecott KW, Ward ME, Nussenzweig A. "Neuron enhancers are a hotspot in DNA single-strand break repair," *Nature*, May 2021; Vol. 593 (Issue 7859): 440-444. doi:

[0264] Vilenchik MM, Knudson AG. "Endogenous DNA Double-Strand Breaks: Precision of Generation, Repair, and Cancer Induction." *Proceedings of the National Academy of Sciences*, October 28, 2003; Vol. 100 (No. 22): 12871-12876. doi: 10.1073 / pnas.2135498100. Electronic publication date: October 17, 2003. PMID: 14566050; PMCID: PMC240711

[0265] Cao et al., Nature Communications (2019), Vol. 10, p. 5799.

[0266] S, Gao Y, Hammarsten O. Numerical analysis of etoposide-induced DNA fragmentation. *PLOS ONE*, June 10, 2009; Vol. 4 (Issue 6): p. e5859. doi: 10.1371 / journal.pone.0005859. Errata in: *PLOS ONE*, 2009; Vol. 4 (Issue 6). doi: 10.1371 / annotation / 290cebfd-d5dc-4bd2-99b4-f4cf0be6c838. PMID: 19516899; PMCID: PMC2689654.

[0267] Baranello et al., *International Journal of Molecular Sciences* (2014), Vol. 15: 13111-1322

[0268] Baranello, L. et al. (2018). “Locating DNA breaks using next-generation sequencing” in: Muzi Falconi, M., Brown, G. (eds.), *Genomic Instability*, *Methods in Molecular Biology*, Vol. 1672, HuMana Publishing, New York, NY.

[0269] https: / / doi.org / 10.1007 / 978-1-4939-7306-4_13

[0270] Hu et al., Genes & Development, 2015, Vol. 29: pp. 948-960.

[0271] Sriramachandran et al., *Molecular Cell* (2020), Vol. 78, pp. 975-985.

[0272] Ewels, PA, Peltzer, A., Fillinger, S., et al., Nature Biotechnology, pp. 38276–38278 (2020).

[0273] https: / / doi.org / 10.1038 / s41587-020-0439-x

[0274] Andrews, S. (2010). FastQC: A quality control tool for high-throughput sequencing data [online]. Available online: http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc /

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[0277] MARTIN, Marcel. Cutadapt: ​​Deleting adapter sequences from high-throughput sequencing reads. EMBnet.journal, [Sl], Vol. 17, No. 1, pp. 10-12, May 2011. ISSN 2226-6089. doi: https: / / doi.org / 10.14806 / ej.17.1.200.

[0278] Heng Li, Richard Durbin, “Efficient and Accurate Long Read Alignment Based on Burrows-Wheeler Transform”, *Bioinformatics*, Vol. 26, No. 5, March 2010, pp. 589-595, https: / / doi.org / 10.1093 / bioinformatics / btp698

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[0281] Table 4: Sequences

Claims

1. A method of detecting a single-strand break (SSB) site in double-stranded (ds) DNA comprising; (i) contacting a dsDNA sample with an oligonucleotide probe comprising a first sequencing adaptor and a tag, (ii) contacting the dsDNA sample with a DNA ligase, such that the DNA ligase covalently ligates the oligonucleotide probe to a strand of the dsDNA at the dsDNA single-strand break site, (iii) contacting the dsDNA sample with a transposase loaded with a second sequencing adaptor, such that the transposase cleaves the dsDNA to generate a population of fragments comprising terminal second sequencing adaptors, (iv) isolating from the population DNA strands comprising first and second sequencing adaptors; (v) determining the sequence of the isolated DNA strands.

2. The method of claim 1, wherein the sequence of the isolated DNA strands is indicative of a single-strand break (SSB) site within the dsDNA.

3. The method of any preceding claim, wherein the dsDNA is genomic DNA.

4. The method of any preceding claim, wherein the dsDNA is in a cell nucleus or extract thereof.

5. The method of any preceding claim, wherein the dsDNA is from a diseased cell; an embryonic cell; an age-related cell; or a treated cell.

6. The method of claim 5, wherein the treated cell has been treated with; exposure to one or more compounds, exposure to light or radiation, exposure to cell culture conditions, exposure to a nuclease, optionally Cas9 or Cpfl, or exposure to reprogramming factors, optionally Oct3 / 4, Sox2, Klf4 and c-Myc.

7. The method of any preceding claim, wherein the dsDNA sample is prepared by a method comprising extraction of cell nuclei from a eukaryotic cell.

8. The method of claim 7, wherein the eukaryotic cell is fixed and permeabilised.

9. The method of any preceding claim, wherein prior to contacting the sample with the oligonucleotide probe, the dsDNA sample can comprise one or more of the following steps; (i) washing and lysing a sample (e.g. a cell) comprising dsDNA, (ii) buffer exchange of the sample comprising dsDNA, for example by centrifugation or dilution of the sample.

10. The method of any preceding claim, wherein following contacting the dsDNA with the ligase, the dsDNA sample is purified by protease digestion.

11. The method of any preceding claim, wherein the isolation of DNA strands comprising first and second sequencing adaptors is performed using magnetic beads.

12. The method of any preceding claim, wherein the tag is biotin.

13. The method of any preceding claim, wherein the probe further comprises a hybridisation region which anneals to the dsDNA at the SSB site.

14. The method of claim 13, the hybridisation sequence comprising 5-10 nucleotides.

15. The method according to any one of claims 13 or 14, wherein the hybridization sequence is diverse.

16. The method of claim 15, wherein the hybridization sequence is random.

17. The method according to any one of claims 13 or 14, wherein the hybridization sequence is complementary to the target sequence in the dsDNA.

18. The method of claim 14, wherein the target sequence is a promoter.

19. The method according to any of the preceding claims, wherein the oligonucleotide probe is attached to the 3' end of the dsDNA break strand at the SSB site.

20. The method of claim 19, wherein the tag is located at the 3' end of the oligonucleotide probe, and the oligonucleotide probe further comprises a 5' phosphate group.

21. The method according to any one of claims 1 to 18, wherein the oligonucleotide probe is attached to the 5' end of the dsDNA break strand at the SSB site.

22. The method of claim 21, wherein the tag is located at the 5' end of the oligonucleotide probe, and the oligonucleotide probe further comprises a 3'-OH group.

23. The method according to any one of the preceding claims, wherein the transposase is a Tn5 transposase.

24. The method according to any one of the preceding claims, wherein the fragments comprising the first and second sequencing adapters are separated by contacting the tag with a fixed binding member.

25. The method of claim 24, wherein the tag is biotin and the binding member is streptavidin.

26. The method according to claim 24 or 25, wherein the connecting member is fixed to the bead.

27. The method according to any one of the preceding claims, comprising treating the isolated DNA strands with bisulfite.

28. The method of claim 27, wherein the sequence of the bisulfite-treated strand is compared with a reference genome to identify methylated cytosine residues in the isolated DNA strand.

29. The method according to any one of the preceding claims, comprising amplifying isolated DNA strands using sequencing primers to produce amplification products.

30. The method according to any one of the preceding claims, comprising sequencing the isolated DNA strands or amplification products.

31. The method according to any one of the preceding claims further includes generating a set of sequence reads of isolated DNA strands or amplification products.

32. The method of claim 31, further comprising locating the sequence reads in the group to one or more sites in a reference genome.

33. The method of claim 32, further comprising determining the frequency of SSBs at said site by the number of sequence reads located to a site in a reference genome.

34. The method of claim 33, further comprising locating the SSB frequencies of sites within the first dsDNA and the second dsDNA, and identifying sites in the first dsDNA where the SSB frequency is increased relative to that in the second dsDNA, or in the second dsDNA where the SSB frequency is increased relative to that in the first dsDNA.

35. The method of claim 34, wherein the first dsDNA is a test sample and the second dsDNA is a reference or control sample.

36. The method of claim 35, wherein the first dsDNA is derived from diseased cells; embryonic cells; age-related cells; or treated cells.

37. The method of claim 36, wherein the treated cells have been treated with a selection from the group consisting of; Exposure to one or more compounds, Exposure to light or radiation, Exposure to cell culture conditions, Exposure to nucleases, optionally Cas9 or Cpf1, or Exposure to Oct3 / 4, Sox2, Klf4 and c-Myc.

38. A kit for detecting single-strand break (SSB) sites in double-stranded (ds) DNA; said kit comprising: Oligonucleotide probes, which contain a first sequencing adapter and a tag, DNA ligase, transposases and A second sequencing adapter loaded with or loadable onto a transposase.

39. The kit according to claim 38, comprising instructions for use according to any one of claims 1 to 37.

40. A method for screening reagents capable of repairing SSBs in dsDNA, such as chemical and / or biological reagents (e.g., small molecule and / or peptide and / or protein therapeutic agents), wherein the screening method comprises the following steps; (a) Detect single-strand break (SSB) sites in double-stranded (ds) DNA control samples that were not exposed to the reagent by the following steps, and detect single-strand break (SSB) sites in test versions of the same dsDNA samples that were exposed to the reagent. (i) Contact the test and control dsDNA samples with an oligonucleotide probe containing a first sequencing adapter and a tag. (ii) Contact the test and control dsDNA samples with DNA ligase, allowing the DNA ligase to covalently link the oligonucleotide probe to the dsDNA strand at the single-strand break site of the dsDNA. (iii) Contact the test and control dsDNA samples with a transposase loaded with a second sequencing adapter, causing the transposase to cleave the dsDNA to produce a fragment cluster containing a terminal second sequencing adapter, and then... (b) Isolate DNA strands containing the first and second sequencing adapters from the group. (c) Determine the sequences of isolated DNA strands from test and control samples. (d) Compare the SSB spectra in the test sample and the control sample. (e) Isolation of reagents (e.g., chemical and / or biological reagents) associated with a reduction in SSB in the test sample compared to the control sample, and (f) The separating reagent obtained from (e) is formulated with a pharmaceutically acceptable excipient.

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