Method and composition for detecting and analyzing nucleic acid molecules

Abstract

The present application relates to a method for analyzing nucleic acid molecules in a sample, the method comprising: a) contacting at least one nucleic acid molecule in a sample with endonuclease to obtain one or more nucleic acid fragments; the present invention relates to a method for determining the sequence and / or modification status of a nucleic acid molecule in a sample, b) contacting one or more nucleic acid fragments with at least one barcoded probe to obtain a barcoded nucleic acid fragment, c) determining the composition of the barcoded nucleic acid fragment and determining the sequence and / or modification status of the nucleic acid molecule in the sample from the composition of the barcoded nucleic acid fragment. The invention also provides a kit comprising the endonuclease and the barcoded probe, and a device for performing the method.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method and composition for analyzing nucleic acids in a sample. Background Art

[0002] Currently, single-cell sequencing is an advanced technology that aims to deeply explore the whole genome or partial genome information of a single cell. Compared with traditional sequencing methods, this technology is expected to provide more detailed and comprehensive genetic information and reveal genetic heterogeneity at the cellular level.

[0003] Single-cell genome sequencing has wide applications in cancer research, developmental biology, genetics and other fields. By analyzing the genome of a single cell, people can gain a deeper understanding of cell mutations, evolutionary processes and genetic diversity. However, there are still many difficulties and challenges. For example, the amount of DNA in a single cell is small and may be limited. For traditional DNA sequencing methods, this means that bias is easily introduced during sample processing and sequencing. In addition, single-cell whole genome DNA detection requires high coverage of the entire genome, and achieving this goal at the cellular level remains a challenge.

[0004] Despite a series of challenges, significant progress has been made in the field of single-cell whole genome sequencing in recent years. Next-generation sequencing technology, improved experimental processes, and advanced data analysis methods have gradually solved some technical difficulties and improved the feasibility and accuracy of the technology.

[0005] In summary, single-cell technology used for DNA monitoring and analysis provides a powerful tool for in-depth understanding of the complexity and heterogeneity of cellular genomes. However, continuous efforts are still needed to address technical challenges, find new sequencing methods or improved experimental processes to improve the sensitivity and accuracy of detection and promote the development of this field. Summary of the invention

[0006] The method of the present application utilizes the characteristics of endonuclease specific recognition of nucleic acid sites and cutting nucleic acid molecules into different fragments, combined with the high-throughput characteristics of single-cell sequencing, to provide a simple, efficient and accurate method for analyzing nucleic acid molecules. Specifically, first, a cell sample or an organelle sample containing nucleic acid can be separated into a single cell or a single organelle, and the nucleic acid molecules (e.g., genomic DNA) therein can be exposed, and then one or more endonucleases are used for cutting, and the longer nucleic acid molecules are processed into DNA fragments with different cuts, and the cut information from the DNA fragments of a single cell is obtained by single-cell sequencing. When combined with high-throughput analysis, the state of the nucleic acid molecules in the sample can be obtained. For example, if a sequence-specific endonuclease is used, the sequence information of the nucleic acid molecule can be obtained by the sequence information at / near the cut. For example, if a modification-dependent endonuclease is used, the modification information of the corresponding position of the nucleic acid molecule can be obtained according to the sequence information at / near the cut obtained. For example, when two or more endonucleases are used, multiple information of multiple cuts can be obtained, and under high-throughput analysis, the sequence and modification complete information of the nucleic acid molecule can be obtained.

[0007] On the one hand, the present application provides a method for analyzing nucleic acid molecules in a sample, the method comprising: a) contacting (at least one) nucleic acid molecule in the sample with a nuclease to obtain one or more nucleic acid fragments; b) contacting the one or more nucleic acid fragments with at least one barcoded probe to obtain barcoded nucleic acid fragments; c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification state of the nucleic acid molecules in the sample from the composition of the barcoded nucleic acid fragments.

[0008] In some embodiments, the method comprises treating nucleic acid molecules in a sample with the endonuclease to obtain one or more nucleic acid fragments with specific sequences at the ends, wherein the nucleic acid fragments are connected to the barcoded probe via the specific sequences at the ends. In some embodiments, the barcoded probe comprises a sequence that hybridizes with the specific sequence at the ends.

[0009] In certain embodiments, the sample is from a cell. In certain embodiments, the sample comprises at least one cell.

[0010] In certain embodiments, the sample is a single cell.

[0011] In certain embodiments, the nucleic acid molecule in the sample is DNA from cells.

[0012] In certain embodiments, the nucleic acid molecules in the sample are one or more DNA molecules in a single cell.

[0013] In some embodiments, the cell is selected from the group consisting of cells isolated from an organism, cells cultured in vitro, primary cells, and discrete cells from an explant. In some embodiments, the cell is derived from a eukaryotic organism. In some embodiments, the cell is derived from a prokaryotic organism. In some embodiments, the cell is derived from a bacterium.

[0014] In certain embodiments, the cells are fixed.

[0015] In certain embodiments, the method comprises separating the sample into single cells. Methods for separation into single cells are known in the art and may include, but are not limited to, micromanipulation, LCM, FACS, and microfluidics.

[0016] In certain embodiments, the sample includes at least one organelle.

[0017] In certain embodiments, the organelles include a nucleus, a chloroplast, and / or a mitochondria.

[0018] In certain embodiments, the sample is a single cell nucleus.

[0019] In certain embodiments, the nucleic acid molecules in the sample are DNA from a single cell nucleus.

[0020] In certain embodiments, the nucleic acid molecules in the sample are one or more DNAs in a single cell nucleus.

[0021] In some embodiments, the cell nucleus is derived from a cell selected from the group consisting of a cell isolated from an organism, a cell cultured in vitro, a primary cell, and a discrete cell from an explant. In some embodiments, the cell is derived from a eukaryotic organism. In some embodiments, the cell is derived from a prokaryotic organism. In some embodiments, the cell is derived from a bacterium.

[0022] In certain embodiments, the method comprises exposing nuclei of cells of the sample.

[0023] In certain embodiments, the method comprises isolating the sample into single cell nuclei.

[0024] In some embodiments, the method comprises exposing the nucleic acid molecule from the sample. In some embodiments, the exposure comprises using a protease. The protease can hydrolyze proteins in the cell, exposing the nucleic acid molecule.

[0025] In certain embodiments, the method comprises partitioning the nucleic acid molecules, the endonucleases, and the barcoded probes into discrete partitions.

[0026] In certain embodiments, the discrete partitions are wells or droplets.

[0027] In certain embodiments, the discrete partitions further comprise a ligase.

[0028] In certain embodiments, the method includes providing a solid support having at least one barcoded probe attached thereto.

[0029] In certain embodiments, the solid support is a bead. In certain embodiments, the bead is a magnetic bead.

[0030] In certain embodiments, the barcoded probes comprise a barcode sequence, and the barcode sequences comprised by the barcoded probes attached to the same solid support are identical.

[0031] In certain embodiments, the barcode sequence comprises at least two cellular barcode segments separated by a linker sequence.

[0032] In certain embodiments, the barcoded probes comprise a universal priming sequence.

[0033] In some embodiments, the barcoded probe comprises a nucleic acid binding molecule. In some embodiments, the nucleic acid binding molecule can be linked to the one or more nucleic acid fragments, for example, by hybridization complementation.

[0034] In certain embodiments, the one or more nucleic acid fragments are bound to the barcoded probe via the nucleic acid binding molecule.

[0035] In certain embodiments, the nucleic acid binding molecule hybridizes to the barcoded probe.

[0036] In certain embodiments, the barcoded probe further comprises a unique molecular identification region.

[0037] In certain embodiments, the method comprises attaching a unique molecular identification region to the one or more nucleic acid fragments.

[0038] In certain embodiments, the endonuclease comprises a Class I restriction endonuclease, a Class II restriction endonuclease, a Class III restriction endonuclease, and a Class IV restriction endonuclease.

[0039] In certain embodiments, the endonuclease comprises a modification-dependent endonuclease. In certain embodiments, the modification-dependent endonuclease comprises a methylation-sensitive endonuclease.

[0040] In some embodiments, the endonuclease comprises two or more endonucleases. In some embodiments, the two or more endonucleases recognize or cut different nucleic acid sites.

[0041] In certain embodiments, the nuclease is selected from any one or more of the following groups: MspJI, AbaSI, BamHI, ClaI, EooRI, HindIII, HindII, KpnI, NotI, PstI, SalI, Sau3AI, SfiI, SmaI, XbaI and XhoI.

[0042] In some embodiments, the method comprises, before step a), pre-treating the nucleic acid molecule. In some embodiments, the pre-treating comprises treating the nucleic acid molecule into a form that can be recognized and cleaved by an endonuclease.

[0043] In certain embodiments, the pretreatment comprises the use of a glycosyltransferase.

[0044] In certain embodiments, the method comprises obtaining the barcoded nucleic acid fragments from discrete partitions.

[0045] In certain embodiments, the method comprises amplifying the barcoded nucleic acid fragments.

[0046] In certain embodiments, the method comprises sequencing the barcoded nucleic acid fragments to thereby determine the composition of the barcoded nucleic acid fragments.

[0047] In certain embodiments, the method includes determining the sequence and / or modification state of the nucleic acid molecule by the number of barcoded nucleic acid fragments after determining the composition of the barcoded probe.

[0048] On the other hand, the present application provides a kit, which comprises the nuclease described in the present application and the barcoded probe described in the present application.

[0049] In certain embodiments, the kit includes at least one of a nucleic acid amplification agent, a linking agent, a fixing agent, a permeabilizing agent, and a lysing agent.

[0050] In certain embodiments, the kit comprises instructions for use.

[0051] In some embodiments, the instructions for use include the following steps: a) contacting at least one nucleic acid molecule in a sample with a nuclease to obtain one or more nucleic acid fragments; b) contacting one or more nucleic acid fragments with at least one barcoded probe to obtain barcoded nucleic acid fragments; c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification state of the nucleic acid molecules in the sample from the composition of the barcoded nucleic acid fragments.

[0052] In another aspect, the present application provides a reagent combination, comprising (1) an endonuclease and (2) a barcoding probe.

[0053] In certain embodiments, the (1) endonuclease and the (2) barcoding probe exist independently of each other.

[0054] In certain embodiments, the endonuclease comprises a Class I restriction endonuclease, a Class II restriction endonuclease, a Class III restriction endonuclease, and a Class IV restriction endonuclease.

[0055] In certain embodiments, the endonuclease comprises a modification-dependent endonuclease.

[0056] In some embodiments, the endonuclease comprises two or more endonucleases. In some embodiments, the two or more endonucleases recognize or cut different nucleic acid sites.

[0057] In certain embodiments, the nuclease is selected from any one or more of the following groups: MspJI, AbaSI, BamHI, ClaI, EooRI, HindIII, HindII, KpnI, NotI, PstI, SalI, Sau3AI, SfiI, SmaI, XbaI and XhoI.

[0058] In certain embodiments, the barcoded probes are attached to a solid support.

[0059] In certain embodiments, the solid support is a bead. In certain embodiments, the bead is a magnetic bead.

[0060] In certain embodiments, the barcoded probes comprise a barcode sequence, and the barcode sequences comprised by the barcoded probes attached to the same solid support are identical.

[0061] In certain embodiments, the barcode sequence comprises at least two cellular barcode segments separated by a linker sequence.

[0062] In certain embodiments, the barcoded probes comprise a universal priming sequence.

[0063] In certain embodiments, the barcoded probe comprises a nucleic acid binding molecule.

[0064] In certain embodiments, the barcoded probe further comprises a unique molecular identification region.

[0065] On the other hand, the present application provides a system for analyzing nucleic acid molecules, wherein the system comprises the endonuclease described in the present application and the barcoded probe described in the present application.

[0066] On the other hand, the present application provides an apparatus for executing the method described in the present application.

[0067] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0069] Figure 1 Shown are exemplary applications of the method described in the present application: encapsulating single cells or cell nuclei into hydrogel beads using a droplet-generating microfluidic device; generating beads with attached barcoded probes by a droplet-generating microfluidic device; and fusing beads containing scDNA and beads containing barcode sequences by another droplet fusion device.

[0070] Figure 2 Shown are droplets containing cell spheroids

[0071] Figure 3 Shown are droplets of cell-microspheres and barcoded microspheres.

[0072] Figure 4 Shown are the results of using the method of the present application to detect the omics information of single cells in mixed cell line samples from different species.

[0073] Figure 5 Shown are the results of using the method of the present application to detect methylation information of single cells in a mixed cell line sample of different human cells.

[0074] Figure 6 Shown is the bulk methylation information of a mixed cell line sample of different human cells.

[0075] Figure 7 Shown is the genomic distribution of characteristic differentially methylated regions in a mixed cell line sample of different human cells.

[0076] Figure 8 Shown are the results of using the method of the present application to detect the methylation information of single cells in a bone marrow mononuclear cell sample. DETAILED DESCRIPTION

[0077] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0078] Definition of terms

[0079] In this application, the term "endonuclease" generally refers to a class of enzymes that can recognize and cut a specific position inside a nucleic acid molecule (DNA or RNA). These enzymes are usually highly specific and only work on a specific nucleic acid sequence or structure. Endonucleases can distinguish specific sequences in target nucleic acid molecules, and this recognition is usually based on the base pairing rules of nucleic acids. Once the target sequence is identified, the endonuclease will cut the nucleic acid chain at a specific position inside the sequence. This cutting activity helps to achieve accurate rupture of specific regions in the genome or RNA molecule. Endonucleases can be divided into restriction endonucleases and non-restriction endonucleases.

[0080] In the present application, the term "barcoded probe" generally refers to a nucleic acid sequence with a unique identification sequence, which can be paired with a target molecule so that the target molecule can be identified and tracked, and generally includes three parts: 1. A part used for PCR amplification, for example, a PCR primer, or a PCR handle, which can be referred to as a "universal priming sequence" in the present application; 2. A sequence used to distinguish molecules from different cells, different molecules or sequencing reads, for example, a cell barcode or a feature barcode, which can be referred to as a "barcode sequence" in the present application, and 3. A part used to bind to a target molecule, for example, a capture, which can be referred to as a "nucleic acid binding molecule" in the present application. The barcoded probe may include one or more barcodes, one or more PCR handles, and one or more captures. The barcodes related to single-cell sequencing in the prior art can all be applied to the invention of the present application. It is a known technique in the art to design a suitable barcoded probe comprising the above three parts according to the single-cell sequencing technology or instrument used. The barcoded probe can be releasably attached to a solid support. Common barcoded probe design methods may include but are not limited to: universal priming sequence-barcode sequence-nucleic acid binding molecule. As used herein, "barcode" or "barcode sequence" refers to any unique sequence tag that can be coupled to at least one nucleotide sequence, for example, to later identify the at least one nucleotide sequence. In the present application, the "nucleic acid binding molecule" may include a sequence on the same nucleic acid molecule as the barcode sequence, or may include a sequence that hybridizes with the nucleic acid molecule where the barcode sequence is located, and its function is to connect the target nucleic acid molecule or nucleic acid fragment.

[0081] In the present application, the term "barcode sequence" generally refers to a nucleotide sequence that can be used to identify the source of a target molecule. The source can be, for example, from the same cell or a different cell. In the case where the nucleic acid molecules are derived from a variety of sources, different barcode sequences can be used to mark the nucleic acid molecules in each source so that the source of the sample can be identified. Barcode sequences (also commonly referred to as indexes, tags, etc. - are well known to those skilled in the art. As known in the art and as exemplified by the disclosures of U.S. Patent No. 8,053,192 and PCT Publication WO 05 / 068656, any suitable barcode sequence or barcode sequence group can be used, and the reference is incorporated herein by reference in its entirety. Barcode sequencing of single cells can be performed as described, for example, in the disclosure of US2013 / 0274117, and the document is incorporated herein by reference in its entirety.

[0082] In this application, the term "solid support" generally refers to any material that is suitable or can be modified to be suitable for attaching oligonucleotide tags, sample-specific barcodes, primers, etc. described herein. For example, the solid support includes an array of holes or depressions located in the surface, which can be manufactured using a variety of techniques, such as photolithography, stamping technology, molding technology, and micro-etching technology; the composition and geometry of the solid support can be changed according to its use, for example, the solid support can be a planar structure (such as a slide, chip, microchip and / or array, etc.); for example, the solid support or its surface can also be non-planar, such as the inner or outer surface of a tube or container; for example, the solid support can also include microspheres or beads.

[0083] In the present application, the term "discrete partition" generally refers to a spatial unit that is independent of each other and contains a target substance to be analyzed. For example, a droplet or a well; for example, a droplet formed by co-distributing the probe with a solid support to which a universal initiation sequence is attached; for example, a droplet formed by co-distributing the probe with a solid support to which an oligonucleotide tag is attached; for example, the probe and the solid support to which a universal initiation sequence is attached are co-distributed into a well of a 96-well plate; for example, the probe and the solid support to which an oligonucleotide tag is attached are co-distributed into a well of a 96-well plate. In some embodiments, the discrete partition may also contain other substances that are distributed according to different needs, such as dyes, emulsifiers, surfactants, stabilizers, polymers, aptamers, reducing agents, initiators, biotin markers, fluorophores, buffers, acidic solutions, alkaline solutions, light-sensitive enzymes, pH-sensitive enzymes, aqueous buffers, detergents, ionic detergents, non-ionic detergents, and the like.

[0084] In this application, the term "beads" or "microspheres" or "particles" generally refers to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex or cross-linked dextran (such as agarose), cellulose, nylon, cross-linked micelles and Teflon, and any other materials for solid supports outlined herein can all be used, and reference can be made to the Microsphere Detection Guide of the Fisher Bangs Laboratories (Bangs Laboratories, Fishers Ind.); in certain embodiments, the microspheres can be magnetic microspheres or beads.

[0085] In the present application, the term "unique molecular identification region" may also be referred to as "molecular barcode", "molecular tag", "unique identifier (UID)", "unique molecular identifier (UMI)", etc., and generally refers to a unique sequence code for each probe connection. It can generally be designed as a completely random nucleotide chain (e.g., NNNNNNN), a partially degenerate nucleotide chain (e.g., NNNRNYN), or a specified nucleotide chain (e.g., when the template molecule is finite). The design, incorporation, and application of UMI can be performed according to methods known in the art, for example, as disclosed in WO2012 / 142213, Islam et al. (Nat. Methods) (2014) 11: 163-166, and Kivioja, T. et al. (Nat. Methods) (2012) 9: 72-74, which are incorporated herein by reference in their entirety.

[0086] In the present application, the term "reagent" generally refers to a reagent or a mixture of two or more reagents that can be used to react with a sample, interact with a sample, dilute a sample, or add a sample, and may include reagents used in the assays described herein, including reagents for cleavage, nucleic acid analysis, nucleic acid amplification reactions, protein analysis, fragment labeling reactions, ATAC-seq, CPT-seq or SCI-seq reactions or other assays. Therefore, reagents may include, for example, buffers, chemicals, enzymes, polymerases, primers with a size of less than 50 base pairs, template nucleic acids, nucleotides, markers, dyes or nucleases. In some embodiments, reagents include lysozyme, proteinase K, random hexamer, polymerases (e.g., DNA polymerases, Taq polymerases, Bsu polymerases), transposases (e.g., Tn5), primers (e.g., P5 and P7 adapter sequences), ligases, catalytic enzymes, deoxynucleotide triphosphates, buffers or divalent cations.

[0087] In this application, unless otherwise indicated, the terms "isolate", "purify" and their grammatical equivalents as used herein refer to the reduction of the amount of at least one contaminant (such as a protein and / or nucleic acid sequence) from a sample or source (e.g., a cell) from which the material is isolated. Thus, purification results in "enrichment", e.g., an increase in the amount of a desired protein and / or nucleic acid sequence in a sample.

[0088] In this application, the term "comprising" generally indicates that a range or element includes other elements or features. This is used to define the scope of a particular technology to make it more flexible and inclusive. When the word "comprising" is used, it means that the solution includes not only the elements explicitly listed, but also other related elements or features to achieve similar functions.

[0089] In this application, the term "and / or" is generally used to indicate a selection range, indicating an inclusive relationship between two or more elements or conditions. When "and / or" is used, it allows one of the elements or conditions, or both, to exist to achieve the same technical effect. For example, if the description contains a statement such as "A and / or B", it means that it can include A or B, or both A and B. DETAILED DESCRIPTION OF THE INVENTION

[0091] Methods and kits

[0092] On the one hand, the present application provides a method for analyzing nucleic acid molecules in a sample, the method comprising: a) contacting at least one nucleic acid molecule in the sample with a nuclease to obtain one or more nucleic acid fragments; b) contacting one or more nucleic acid fragments with at least one barcoded probe to obtain barcoded nucleic acid fragments; c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification state of the nucleic acid molecules in the sample from the composition of the barcoded nucleic acid fragments.

[0093] In another aspect, the present application provides a reagent combination for analyzing nucleic acid molecules in a sample, the reagent combination comprising (1) an endonuclease and (2) a barcoding probe.

[0094] On the other hand, the present application provides a kit, which comprises (1) a nuclease, and (2) a barcoded probe. The kit may also include at least one of a nucleic acid amplifier, a linker, a fixative, a permeabilizer, and a lysing agent. The kit may also include instructions for use. The instructions for use may include the following steps: a) contacting at least one nucleic acid molecule in a sample with a nuclease to obtain one or more nucleic acid fragments; b) contacting one or more nucleic acid fragments with at least one barcoded probe to obtain barcoded nucleic acid fragments, c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification state of the nucleic acid molecules in the sample from the composition of the barcoded nucleic acid fragments.

[0095] sample

[0096] In the present application, the sample can be derived from almost any organism, and can include multicellular organisms, such as plants, fungi, bacteria and animals. For example, the sample can be derived from animals (e.g., mammals). For example, the sample can be derived from humans. For example, the sample can be a biopsy tumor or a portion thereof. For example, the sample can be a clinically relevant tissue sample. For example, the tumor can include a solid tumor. For example, the tumor can include a hematological tumor. For example, the sample can be derived from a mixture of a tumor cell line and a non-tumor cell line. For example, the sample can be derived from a mixed cell line of a 293T cell line, a 3T3 cell line, a Hela cell line and / or a Jurkat cell line. For example, the sample can be from a tumor tissue. For example, the sample can be from a bone marrow cell (e.g., a bone marrow mononuclear cell).

[0097] In the present application, a sample may comprise any number of substances, including but not limited to: cells of virtually any organism (including both primary cells and cultured cell lines), cell lysates or extracts (including but not limited to RNA extracts, purified mRNA), tissues and tissue extracts (including but not limited to RNA extracts, purified mRNA), body fluids (including but not limited to blood, urine, serum, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, sweat and semen, exudates, exudates (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis); research samples, including extracellular fluid, extracellular supernatant from cell culture, inclusion bodies in bacteria, cellular compartments, periplasm, mitochondrial compartments.

[0098] In the present application, the sample may include at least one cell. For example, the sample may include one or more, two or more, three or more, four or more, five or more or six or more cells. For example, the sample may be a single cell.

[0099] In the present application, the sample may include an organelle, and the organelle contains genomic information. For example, the sample may be a nucleus. For example, the sample may be a chloroplast. For example, the sample may be a mitochondria.

[0100] In the present application, the sample may include at least one cell nucleus. For example, the sample may include one or more, two or more, three or more, four or more, five or more or six or more cell nuclei. For example, the sample may be a single cell nucleus.

[0101] In the present application, the sample may include at least one mitochondria. For example, the sample may include one or more, two or more, three or more, four or more, five or more or six or more mitochondria. For example, the sample may be a single mitochondria.

[0102] In the present application, the sample may include at least one chloroplast. For example, the sample may include one or more, two or more, three or more, four or more, five or more or six or more chloroplasts. For example, the sample may be a single chloroplast.

[0103] In some embodiments, the method comprises preparing a sequencing library from genomic DNA, nuclear DNA, chloroplast DNA, or mitochondrial DNA from a single cell. The method comprises releasing an organelle from a single cell to provide a plurality of organelles. The organelles are spatially spaced from each other so that one organelle is present in one spatial compartment.

[0104] Preprocessing

[0105] In the present application, before "a) contacting at least one nucleic acid molecule in the sample with a nuclease to obtain one or more nucleic acid fragments", the method may further include exposing the nucleic acid molecule to be detected from the sample. The exposure may refer to separating the nucleic acid molecule to be detected from other components in the sample, such as proteins, lipids, etc. When the nucleic acid molecule is DNA, RNA also needs to be separated. In a cell or organelle sample, nucleic acid molecules coexist with other components. For example, nucleic acid molecules and proteins form chromosomes together, or are mixed with proteins, lipids, carbohydrates, etc. in a space. The nucleic acid molecules can be separated from other components by the "exposure" described in the present application. The separation includes separating the nucleic acid molecules from the sample, separating other components in the sample from the sample, digesting other components in the sample by biochemical methods to leave nucleic acid molecules, etc., as long as a single nucleic acid molecule can be obtained. According to the presence state of the nucleic acid molecules and other components in the sample, those skilled in the art can choose a suitable method to obtain exposed nucleic acid molecules that do not contain other components from the sample.

[0106] In a specific embodiment, proteins present in the sample together with nucleic acid molecules can be digested by protease. The protease that can be used for "exposing" can be a serine protease, for example, proteinase K.

[0107] Endonuclease

[0108] Endonucleases can be a biomolecule or enzyme that has the ability to recognize a specific sequence on a nucleic acid (e.g., DNA or RNA) chain and cut nucleic acids at or near the sequence. Endonucleases used for the present application can be restriction endonucleases that can recognize specific DNA or RNA sequences to destroy the phosphodiester bonds in a polynucleotide chain.

[0109] In some embodiments, one or more restriction endonucleases are contacted with the exposed nucleic acid molecules, wherein one restriction endonuclease can cut the genomic nucleic acid molecules into nucleic acid molecule fragments having the same sticky ends or blunt ends, and multiple restriction endonucleases can cut the genomic nucleic acid molecules into nucleic acid molecule fragments having multiple sticky ends or blunt ends.

[0110] In other embodiments, the endonuclease is modification-dependent, i.e., recognizes nucleotide modifications in nucleic acid molecules. Common modification-dependent endonucleases include AbaSI from Acinetobacter, which can recognize 5-glucosylhydroxymethylcytosine (5 ghm C) or 5-hydroxymethylcytosine (5 hmC), also includes MspJI from mycobacteria, which can be C5-methylated cytosine (5-mC) or C5-hydroxymethylated cytosine (5-mC), further includes PvuRtsl I from Proteus and AspBI from Azoarium, etc., all of which contain an N-terminal nuclease domain and a C-terminal SRA domain, which can recognize methylated or hydroxymethylated DNA (5mC and 5hmC) sites and complete the double-stranded DNA cleavage reaction near the fixed site. Nucleases that can recognize other DNA modifications can also achieve the purpose of the present invention. According to the teachings of the present invention, those skilled in the art can select one or more suitable nucleases based on the information of the nucleic acid molecule to be detected, such as sequence information or modification information.

[0111] AatII, AbaSI, Acc65I, AccII,AcuI,AcI, Aat II, Acc. I,AfeI,AflII,AflIII,AgeI,AhdI,AleI,AluI,AlwI,AlwNI,ApaI,ApaLI,A peKI,ApoI,AscI,AscI,AseI,AsiSI,AvaI,AvaII,AvrII,BaeGI,BaeI,Bam HI,BanI,BanII,BbsI,BbvCI,BbvI,BccI,BceAI,BcgI,BciVI,BclI,BcoDI, BfaI,BfuAI,BfuCI,BglI,BglII,BlpI,BmgBI,BmrI,BmtI,BpmI,Bpu10I,B puEI,BsaAI,BsaBI,BsaHI,BsaI,BsaJI,BsaWI,BsaXI,BseRI,BseYI,BsgI, BsiEI,BsiHKAI,BsiWI,BslI,BsmAI,BsmBI,BsmFI,BsmI,BsoBI,Bsp1286I ,BspCNI,BspDI,BspEI,BspHI,BspMI,BspQI,BsrBI,BsrDI,BsrFI,BsprGI,B srI,BssHII,BssKI,BssSI,BstAPI,BstBI,BstEII,BstNI,BstUI,BstXI,B stYI,BstZ17I,Bsu36I,BtgI,BtgZI,BtsCI,BtsI,BtsIMutI,Cac8I,ClaI,C spCI,CviAII,CviKI-1,CviQI,DdeI,DpnI,DpnII,DraI,DraIII,DrdI,Eae I,EagI,EarI,EciI,Eco53kI,EcoNI,EcoO109I,EcoP15I,EcoRI,EcoRV,Fat I,FauI,Fnu4HI,FokI,FseI,FspEI,FspI,HaeII,HaeIII,HgaI,HhaI,Hinc II,HindIII,HinfI,HinP1I,HpaI,HpaII,HphI,Hpy166II,Hpy188I,Hpy188 III,Hpy99I,HpyAV,HpyCH4III,HpyCH4IV,HpyCH4V,I-CeuI,I-SceI,KasI, KpnI,LpnPI,MboI,MboII,MfeI,MluCI,MluI,MlyI,MmeI,MnlI,MscI,MseI,MslI,MspA1I,MspI,MspJI,MwoI,NaeI,NarI,Nb.BbvCI,Nb.BsmI,Nb.Bsr DI,Nb.BtsI,NciI,NcoI,NdeI,NgoMIV,NheI,NlaIII,NlaIV,NmeAIII,Not I,NruI,NsiI,NspI,Nt.AlwI,Nt.BbvCI,Nt.BsmAI,Nt.BspQI,Nt.BstNBI, Nt.CviPII,PacI,PaeR7I,PciI,PflFI,PflMI,PhoI,PI-PspI,PI-SceI,Pl eI,PluTI,PmeI,PmlI,PpuMI,PshAI,PsiI,PspGI,PspOMI,PspXI,PstI,P VuI,PvuII,RsaI,RsrII,SacI,SacII,SalI,SapI,Sau3AI,Sau96I,SbfI,S caI,ScrFI,SexAI,SfaNI,SfcI,SfiI,SfoI,SgrAI,SmaI,SmlI,SnaBI,Spe I,Sphl,SspI,StuI,StyD4I,StyI,SwaI,TaqαI,Tfil,TliI,TseI,Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, ZraI, EcoRI,EcoRII,BamHI,HindIII,TaqI,NotI,HinfI,Sau3A,PovII,SmaI ,HaeIII,HgaI,AluI,EcoRV,EcoP15I,KpnI,PstI,SacI,SalI,ScaI,SphI, StuI,XbaI,AarI,BanII,BseGI,BspPI,CfrI,EcoNI,Hsp92II,NlaIV,RsaI ,TaiI,AasI,BbsI,BseJI,BspTI,ClaI,I-PpoI,NmuCI,RsrII,TaqaI,Aat II,BbuI,BseLI,BsrBI,CpoI,KasI,Acc65I,BbvCI,BseMI,BsrDI,Csp45I, ,Kpn2I,NruI,SacII,TasI,AccB7I,BbvI,BseMII,BsrFI,Csp6I,EheI,Kpn I,NsbI,SalI,TatI,AccI,BceAI,BseNI,BsrGI,CspI,Esp3I,KspAI,NsiI,SapI and TauI. In some embodiments, the endonuclease may include MspjI. In some embodiments, the endonuclease may include AbaSI. In some embodiments, the endonuclease may include different sequence-specific restriction endonucleases.

[0112] In certain embodiments, the endonuclease is a methylation modification-sensitive endonuclease, such as cytosine methylation modification. Cytosine modifications known in the art include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC).

[0113] The recognition site of MspJI is m CNNR: , wherein R is A or G, and Y is C or T, MspJI can generate double-stranded DNA breaks at N9 and / or N13 at the 3' end of the modified cytosine.

[0114] For example, the nucleic acid binding molecule can be designed to recognize or bind to the sequence of the DNA break ends formed after MspJI cleavage, and the barcoded probe can bind to the one or more nucleic acid fragments through the nucleic acid binding molecule to form the barcoded nucleic acid.

[0115] In certain embodiments, a 4-base recognition endonuclease is used, such as an enzyme selected from Mspl, Hpall, Dpnl, and HpyCH4IV.

[0116] In certain embodiments, a 6-base recognition endonuclease is used, such as an enzyme selected from the group consisting of EcoRI, BamI, and XbaI.

[0117] In certain embodiments, one, two, or three endonucleases with a 4-mer recognition sequence and one, two, or three endonucleases with a 6-mer recognition sequence are used.

[0118] In certain embodiments, the endonuclease comprises an enzyme selected from any one of MspI, HpaII, DpnI and HpyCH4IV, and an enzyme selected from any one of EcoRI, BamI and XbaI.

[0119] In certain embodiments, the nucleic acid fragment is 200-800 bp in length.

[0120] In certain embodiments, before "a) contacting at least one nucleic acid molecule in the sample with an endonuclease to obtain one or more nucleic acid fragments", the method may further include treating the nucleic acid molecule into a form that can be recognized and cleaved by the endonuclease.

[0121] In some embodiments, in order to meet the cleavage conditions of some nucleases, the nucleic acid molecules can also be pretreated. For example, in the application of detecting hydroxymethylation (5hmC) of nucleic acid molecules, before using AbaSI enzyme cleavage, the pretreatment can include using galactosyltransferase to treat the nucleic acid molecules.

[0122] The recognition site of AbaSI is ,in, x C is ghm C. hm C. m C or C, AbaSI can recognize 5-glucosylhydroxymethylcytosine (5 ghm C) or 5-hydroxymethylcytosine (5 hm C), and cuts further away from the 3' end of the modified cytosine to form a 2-3 base 3' overhang.

[0123] For example, the nucleic acid binding molecule can be designed to recognize or bind to the sequence of the protruding end formed after AbaSI cleavage, and the barcoded probe can be bound to the one or more nucleic acid fragments through the nucleic acid binding molecule to form the barcoded nucleic acid.

[0124] Discrete partition

[0125] The application provides a device for co-distributing a solid support (e.g., a bead) with a sample, for example, for co-distributing sample components and beads to the same discrete partition. For example, the solid support to which at least one barcoded probe is attached and at least one cell or at least one organelle are co-distributed into the discrete partition. In some embodiments, the solid support is an etched surface, a hole, an array, a flow cell device, a microfluidic channel, a bead, a magnetic bead, a column, a droplet, or a microparticle.

[0126] For example, the discrete partitions may include wells or droplets. For example, the wells may include wells of a cell culture plate or any other container wells that can cooperate with the device and are suitable for co-distribution. For example, the wells may include wells of a 96-well plate. For example, the discrete partitions are droplets. For example, each of the discrete partitions includes at most a probe that binds to the nucleic acid molecule derived from a single cell. For example, the nucleic acid molecule is located in a single cell or a cell nucleus. For example, the nucleic acid molecule that binds to a single cell is co-distributed to the discrete partitions with the solid support to which the barcoded probe is attached using a microfluidic device.

[0127] For example, discrete partitions (e.g., droplets or wells) contain single cells and are processed according to the methods described herein. For example, discrete partitions contain single cells and / or single cell nuclei. Single cells and / or single cell nuclei can be distributed and processed according to the methods described herein. For example, a single cell nucleus can be a component of a cell. For example, discrete partitions contain chromatin (e.g., a single chromosome or other part of a genome) from a single cell or a single cell nucleus and are distributed and processed according to the methods described herein.

[0128] For example, the discrete partitions also include a ligase, and the ligase connects the barcoded probe to the nucleic acid molecule. The discrete partitions include but are not limited to ligases, and may also include other required enzymes. For example, DNA polymerase, DNA endonuclease, DNA exonuclease, terminal transferase, and a light-sensitive enzyme or pH-sensitive enzyme that can release the barcoded probe from the solid support. The ligase includes T4 ligase, but is not limited to T4 ligase, and may also include Escherichia coli DNA ligase, T4 DNA ligase, T7 DNA ligase, mammalian ligase (e.g., DNA ligase I, DNA ligase III, DNA ligase IV), thermostable ligase, etc.

[0129] In the present application, the microfluidic device can be a droplet generator. For example, a solid support to which at least one barcoded probe is attached can be combined with a sample (e.g., a sample containing nucleic acid molecules) by forming an aqueous droplet containing both the solid support to which at least one barcoded probe is attached and the sample. The aqueous droplets serve as discrete partitions. The aqueous droplets can be an aqueous core surrounded by an oil phase, for example, an aqueous droplet in an oil-in-water emulsion. The aqueous droplets can contain one or more solid supports to which at least one barcoded probe is attached, a sample, an amplification reagent, and a reducing agent. In short, the composition of the aqueous droplets will vary according to specific processing requirements.

[0130] The aqueous droplets can have a uniform size or an uneven size. For example, the diameter of the aqueous droplets can be about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm or 1 mm. For example, the fluid droplets can have a diameter of at least about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or more. For example, the fluid droplets can have a diameter of less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 45 μm, 50 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm. For example, the fluid droplets can have a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

[0131] Barcoded probes

[0132] In the present application, barcoding probes can be designed by commonly used methods. For example, barcoding probes can be designed by Oligo 6. For example, barcoding probes can be designed by BLAST. For example, barcoding probes can be designed by Beacon Designer 7.0. For example, barcoding probes can be designed by Primer Express 3.0. For example, barcoding probes can be designed by PrimerPremier 5. For example, barcoding probes can be designed by Primer Designer-M. In the present application, barcoding probes can be designed by Primer3.

[0133] In some embodiments, the barcoded probe is enzymatically generated by a barcode adapter template. A barcode adapter template refers to a nucleic acid molecule containing a barcode sequence that can be used as a template to amplify and manufacture a single-stranded barcoded probe, which can be a double-stranded DNA molecule containing a barcode sequence, and other sequence regions to promote the generation of barcoded probes and subsequent barcoding of nucleic acids. The barcode adapter template can be prepared using standard molecular cloning techniques. In some embodiments, the barcode adapter template includes a promoter for RNA polymerase (RNAP), such as T7, T3 or SP6 promoter. The RNA barcoded probe can then be generated by contacting the template molecule with an appropriate RNAP and allowing in vitro transcription to occur. In some embodiments, the barcode adapter template includes a nicking endonuclease restriction site, such as Nt.BbvCI, Nt.BspQI, Nt.BsmAI, Nt.BstNBI, Nt.AlwI or Nt.BsmAI site. DNA barcoding probes can be produced by contacting the template with a nicking endonuclease specific for the restriction site and then exposing the template to a strand displacement DNA polymerase. Examples of suitable strand displacement DNA polymerases include Klenow exo-fragments, Bst large fragments, and engineered variants thereof. In general, barcoding probes are produced by contacting the template with one or more enzymes from a barcode adapter template. In some embodiments, the enzyme reaction is an isothermal reaction. In some embodiments, barcoding probes can also be synthesized using chemical synthesis or enzyme synthesis of DNA. Any physical, chemical or biological method or combination thereof capable of synthesizing DNA in this area can be used for the synthesis of the barcoding probes of the present invention.

[0134] The barcode adapter template can be in a free form in solution when it is used to generate a barcoded probe, or it can be bound to a solid support. Examples of solid supports that can be used in embodiments of the methods and compositions of the present invention include beads, chromatographic resins, multiwell plates, microcentrifuge tubes, or other objects with a solid surface. The barcode adapter template can be bound to a solid support using any desired mechanism or capture chemistry, such as biotin-avidin, biotin-streptavidin, or gold-thiol interactions. In some embodiments, any solid support connected to the barcode adapter template is contacted with an aqueous solution, and the barcoded probes generated by the template are released into this solution when they are generated. The aqueous solution can be in the same reaction volume as the nucleic acid molecules associated with the sample to which the barcoded probe is to be added. That is, the barcoded probe can be generated in situ for the barcoding reaction. Alternatively, the aqueous solution contacted with the solid support for the barcode adapter template can be contained in a reaction volume different from the target nucleic acid molecules, and the barcoded probes generated from the template can be added to these nucleic acid molecules after combining the two reaction volumes.

[0135] In some embodiments, barcoded probes are generated by cleaving a barcode adapter template from a solid support. The template molecule may contain an endonuclease restriction site that promotes cleavage of the template molecule upon exposure to an appropriate enzyme (e.g., a restriction endonuclease). The nucleic acid molecules released into solution upon cleavage may be used as barcoded probes and directly participate in a barcoding reaction, or may be subjected to further enzymatic reactions (e.g., in vitro transcription) to generate barcoded probes.

[0136] Regardless of how the barcoded probes are generated, libraries of these molecules can be prepared to barcode nucleic acids from a variety of samples. The barcoded probes can be separated into different reaction volumes so that each reaction volume contains, for example, an average of one barcoded probe. Alternatively, each reaction volume can contain multiple copies of a barcoded probe, wherein each copy contains the same barcode sequence. The reaction volumes can be microfluidic droplets or can be enclosed in microcentrifuge tubes or other containers.

[0137] In addition to the barcode sequence, the barcoded probe may also include a universal priming sequence or a universal priming region and a nucleic acid binding molecule. The barcoded probe may also include a unique molecular identification region (UMI). In some implementations, the UMI sequence contains randomized nucleotides and is incorporated into the barcoded probe (or a barcode adapter template that generates the barcoded probe) independently of the barcode sequence. Therefore, a collection of barcoded probes containing the same barcode sequence may contain different UMI sequences. In implementations in which the collection of barcoded probes containing the same barcode sequence but containing different UMI sequences is added to the nucleic acid molecules associated with a sample, each nucleic acid molecule sequence may be connected to a different UMI sequence during barcoding.

[0138] Nucleic acid binding molecules can be molecules that preferentially bind nucleic acids (e.g., bind DNA or RNA). Nucleic acid binding molecules (such as DNA binding molecules) can be specific to a certain type of nucleic acid without binding other types of nucleic acids. For example, DNA binding molecules can preferentially bind DNA, but do not bind or bind other nucleic acids to a lower degree. Examples of nucleic acid binding molecules include dyes or stains, proteins, enzymes, biomolecules, affinity tags, particles, fluorescent markers, peptides, ligands, or other molecules that can specifically bind nucleic acids. Therefore, in some examples, nucleic acid binding molecules are Hoechst dyes, cyanine dyes (including, for example, SYBR dyes, such as SYBR green, SYBR gold, oxazole yellow, thiazole orange, PicoGreen, Safegreen), 4′, 6-diamidino-2-phenylindole (DAPI) or Sytox dyes (including, for example, Sytox green or Sytoxorange). Other nucleic acid binding molecules can include, for example, 7-AAD (7-aminoactinomycin D), acridine orange, acridine red, Alexa Fluor 594, Alexa Fluor 610R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, allophycocyanin (APC), BOBO-3-DNA, BOBO-3, Bodipy650 / 665-X, Cy5.5, Cy5, DDAO, Draq5, ethidium bromide, ethidium bromide azide, ethidium homodimer, ethidium homodimer-1 (EthD-1), ethidium homodimer-1-DNA, ethidium homodimer-2, LDS 751, LDS 751 (DNA), LOLO-1, MitoTrackerred, Nile blue-EtOH, OliGreen, dsDNA quantitative reagent, POPO-1-DNA, PO-PRO-1-DNA, propidium iodide (PI), propidium iodide-DNA, Ribogreen, SYPRO Ruby, SYTO 60, SYTO 61, SYTO62, SYTO 63, SYTO64, Texas Red, TO-PRO-1-DNA, TO-PRO-3, TO-PRO-5, TOTO-1-DNA, TOTO-3, YO-PRO-1-DNA, YO-PRO-3, YOYO-1, YOYO-1-DNA and YOYO-3.These nucleic acid binding molecules are exemplary molecules that can be used, and those skilled in the art will recognize that any nucleic acid binding molecule that distinguishes the target nucleic acid of interest from the off-target nucleic acid can be used. In some embodiments, the DNA binding molecule includes a DNA dye, an affinity tag, a ligand, an enzyme, a peptide, or a biomolecule. In some embodiments, the DNA dye includes Hoechst dye, SYBR Gold, Sytox Orange, PicoGreen, or Qubit.

[0139] The barcoded probe can be an RNA or DNA molecule, or an RNA-DNA hybrid. For example, an adaptor can include an RNA nucleotide covalently linked to a DNA nucleotide in a common oligonucleotide chain. The barcoded probe can also be single-stranded or double-stranded. If double-stranded, the barcoded probe can have one or more blunt ends or ends with single-stranded overhangs.

[0140] In the method of the present application, the barcoded probe may comprise three parts: a universal priming sequence, a barcode sequence and a nucleic acid binding molecule. The universal priming sequence may be operably linked to the barcode sequence. The barcode sequence may be operably linked to the nucleic acid binding molecule.

[0141] For example, the barcoded probe further comprises a linker sequence, and its 5' end can be connected to the solid support through the linker sequence. The linker sequence can include acrydite modification, photocleavage modification, SS modification, dU base modification and other sequences, and can be disconnected by various methods to release the barcoded probe.

[0142] For example, the barcoded probe may further comprise other functional sequences, such as complete or partial functional sequences (e.g., primer sequence (e.g., universal primer sequence, targeted primer sequence, random primer sequence) recognition region, primer annealing sequence, attachment sequence, sequencing primer recognition region, amplification primer recognition region (e.g., universal amplification primer recognition region), etc.) for subsequent processing.

[0143] For example, the subsequent treatment includes amplification. For example, the amplification may include PCR amplification (e.g., Taq DNA polymerase amplification, Super Taq DNA polymerase amplification, LATaq DNA polymerase amplification, Pfu DNA polymerase amplification, Phusion DNA polymerase amplification, KOD DNA polymerase amplification, etc.), isothermal amplification (e.g., loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), etc.), T7 promoter linear amplification, degenerate oligonucleotide primer PCR amplification (DOP-PCR), multiple displacement amplification (MDA), multiple annealing loop amplification technique (MALBAC), etc.

[0144] For example, the cell barcode sequence may not include a linker, and the cell barcode sequence may be a separate nucleic acid sequence synthesized by other methods.

[0145] For example, the universal primer sequence may include P5 or other suitable primers. Universal primers (e.g., P5) may also be compatible with sequencing devices, such as being able to be attached to a flow cell in a sequencing device. For example, such universal primer sequences may provide complementary sequences of oligonucleotides constrained on the surface of a flow cell in a sequencing device, so that a barcoded target nucleic acid sequence may be fixed to the surface for sequencing.

[0146] For example, an amplification primer sequence is a primer sequence used to perform an amplification or replication process (eg, extending a primer along a target nucleic acid sequence) to generate an amplified barcoded probe sequence.

[0147] For example, the sequencing primer sequence, the resulting amplified target sequence will contain such primers and be easily transferred to a sequencing system. For example, when the amplified target is sequenced using an Illumina sequencing system, the sequencing primer sequence may contain a read length 1 (Read1) primer sequence and a read length 2 (Read2) primer sequence.

[0148] For example, the barcoded probe may include a T7 promoter sequence. For example, each cell barcode segment is connected to form a barcoded probe using a ligation reaction. The connection may include joining two nucleic acid segments together by catalyzing the formation of a phosphodiester bond. The ligation reaction may include a DNA ligase, such as Escherichia coli DNA ligase, T4 DNA ligase, mammalian ligase (e.g., DNA ligase I, DNA ligase III, DNA ligase IV), a thermostable ligase, etc. T4 DNA ligase can connect segments containing DNA, oligonucleotides, RNA, and RNA-DNA hybrids. The ligation reaction may not include DNA ligase, but may use alternatives such as topoisomerase. Rapid connection can be achieved by using a high concentration of DNA ligase and including PEG. In order to select a favorable temperature for the ligation reaction, the optimum temperature of the DNA ligase (e.g., 37° C.) and the melting temperature of the DNA to be connected may be considered. The sample and the barcoded solid support may be suspended in a buffer to minimize the ionic effects that may affect the connection.

[0149] The barcoded probe may include one or more functional sequences or components selected from primer sequences, anchor sequences, universal sequences, spacers, index sequences, capture sequences, barcode sequences, cleavage sequences, sequencing-related sequences, and combinations thereof. In some embodiments, the barcoded probe includes a primer sequence. In other embodiments, the barcoded probe includes a primer sequence and a barcode sequence. The primer sequence may also be a universal sequence. The application is not limited to the type of adaptor sequence that can be used, and the technician will recognize that other sequences that can be used for library preparation and next-generation sequencing. Universal sequence is a nucleotide sequence region common to two or more nucleic acid fragments. Optionally, the two or more nucleic acid fragments also have a sequence difference region. Universal sequences that may be present in different members of a plurality of nucleic acid fragments may allow the use of a single universal primer complementary to the universal sequence to replicate or amplify a plurality of different sequences.

[0150] The barcoded probes include nucleic acids, such as single-stranded nucleic acids. The adapters may include short nucleic acids less than, greater than, or equal to about 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides in length, or in a range between any two of the aforementioned sizes.

[0151] For the design and use of barcoded probes, reference may be made to PCT publication WO2017040306A1, which is incorporated herein by reference in its entirety.

[0152] For example, each cellular barcode segment is linked by polymerase chain reaction (PCR) to form a barcoded probe.

[0153] The following are several common barcoded probe designs attached to solid supports, all of which can be used for the purpose of the present invention: 1. bead-T7 promoter-adapter-barcode1-anchor-barcode2-nucleic acid binding molecule

[0154] 2.bead-T7 promoter-adapter-barcode1-anchor-barcode2-UMI-nucleic acid binding molecule

[0155] 3.bead-PCR handle-barcode-UMI-nucleic acid binding molecule

[0156] 4.bead-Seq primer-barcode-UMI-nucleic acid binding molecules

[0157] 5.bead-Seq primer-barcode-nucleic acid binding molecules

[0158] 6.bead-T7 promoter-PCR primer-barcode-UMI-nucleic acid binding molecule

[0159] 7.bead-PCR adaptor-barcode1-fix1-barcode2-fix2-barcode3-nucleic acid binding molecule

[0160] As an example, the barcoded probe design format can be found in PCT International Publication No. WO2022 / 242734A1, which is incorporated herein by reference in its entirety.

[0161] In the present application, the method further comprises obtaining the barcoded nucleic acid fragments from discrete partitions.

[0162] In the present application, the method further comprises amplifying the barcoded nucleic acid fragment.

[0163] In the present application, the method also sequences the barcoded nucleic acid fragments to determine the composition of the barcoded nucleic acid fragments. The term "sequencing" refers to a method by which the identity of at least 10 consecutive nucleotides of a polynucleotide (e.g., the identity of at least 20, at least 50, at least 100 or at least 200 or more consecutive nucleotides) is obtained. In the present application, "sequencing" generally refers to high-throughput sequencing technology, including but not limited to massive parallel sequencing, high-throughput sequencing, connection sequencing (e.g., SOLiD sequencing), proton ion semiconductor sequencing, DNA nanoball sequencing, single molecule sequencing, and nanopore sequencing, and may refer to parallel sequencing synthesis platforms or sequencing connection platforms currently used by Illumina, Life Technologies, or Roche. The next generation sequencing method may also include a nanopore sequencing method or a method based on electronic detection, such as the Ion Torrent technology commercialized by Life Technologies or a method based on single molecule fluorescence commercialized by Pacific Biosciences and / or BGI Microfluidics. Exemplary sequencing technologies include targeted sequencing, single molecule real-time sequencing, electron microscopy-based sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, targeted sequencing, exon sequencing, whole genome sequencing, hybridization sequencing (e.g., in an array such as a microarray), pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel shotgun sequencing, emulsion PCR, low denaturing temperature co-amplification PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, ion semiconductor sequencing, nanoball sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, miSeq (Illumina), HiSeq2000 (Illumina), HiSeq 2500 (Illumina), Illumina Genome Analyzer (Illumina), IonTorrent PGMTM (LifeTechnologies), MinIONTM (Oxford Nanopore Technologies), Real-time SMRTTM technology (PacificBiosciences), Probe Anchor Ligation (cPALTM) (Complete Genomics / BGI), Sequencing, MS-PET sequencing, mass spectrometry, and combinations thereof. In some embodiments, sequencing includes detecting sequencing products using an instrument such as, but not limited to, ABI 377DNA sequencer; ABI 310, 3100, 3100-Avant, 3730, or 373OxI Genetic Analyzer; ABI 3700DNA analyzer; or AppliedBiosystems SOLiDTM system (all from AppliedBiosystems); Genome Sequencer 20 system (Roche Applied Science); or mass spectrometer. In certain embodiments, sequencing includes emulsion PCR. In certain embodiments, sequencing includes high-throughput sequencing technology. In certain embodiments, sequencing includes whole genome sequencing. In certain embodiments, sequencing includes massively parallel sequencing (e.g., massively parallel shotgun sequencing). In alternative embodiments, sequencing includes targeted sequencing.

[0164] In the present application, the method further comprises determining the sequence and / or modification state of the nucleic acid molecule by the number of barcoded nucleic acid fragments after determining the composition of the barcoded probe. According to the sequence and / or modification state of the nucleic acid molecule, the omics information of the sample can be obtained, including genomics, proteomics, and metabolomics, etc. For example, according to the sequence and / or modification state of the nucleic acid molecule, the whole genome information of the sample can be obtained. For example, according to the sequence and / or modification state of the nucleic acid molecule, partial genome information of the sample can be obtained. For example, according to the sequence and / or modification state of the nucleic acid molecule, whether the sample contains methylation modification, or the degree, distribution and / or ratio of methylation modification can be obtained. According to the sequence and / or modification state of the nucleic acid molecule, whether the sample contains epigenetic modification, or the degree, distribution and / or ratio of epigenetic modification can be obtained. For example, according to the sequence and / or modification state of the nucleic acid molecule, the source of the sample can be identified, for example, species, organ or tissue source.

[0165] On the other hand, the present application provides a method for detecting sample omics information.

[0166] On the other hand, the present application provides a method for detecting epigenetic modifications of nucleic acid molecules in a sample.

[0167] On the other hand, the present application provides a method for detecting the source of a sample.

[0168] In certain embodiments, the method may be a method for non-disease diagnosis purposes.

[0169] In other embodiments, the method can be used for disease diagnosis purposes.

[0170] Device

[0171] In another aspect, the present application provides a device for performing the methods described herein. The device can be present on a microfluidic scale and can be phase separated from a carrier fluid. Examples of reaction volumes that can be processed by the device include aqueous droplets in an inverted emulsion (i.e., a water / oil emulsion). The device allows barcoded probes, samples (e.g., cells), biochemical reagents (e.g., enzymes), and / or nucleic acid molecules obtained from these samples to be encapsulated in droplets separately or together.

[0172] In the present application, the device or system comprises a first microfluidic module containing a sample or nucleic acid molecule, a second microfluidic module containing a solid support to which a barcoded probe is attached, and a third module containing an endonuclease. The first microfluidic module may also contain a reagent for cell lysis. The third module may also contain an enzyme or reagent for producing a barcoded probe.

[0173] On the other hand, the present application provides the following embodiments:

[0174] 1. A method for analyzing nucleic acid molecules in a sample, the method comprising:

[0175] a) contacting the nucleic acid molecules in the sample with an endonuclease to obtain one or more nucleic acid fragments;

[0176] b) contacting the one or more nucleic acid fragments with at least one barcoding probe to obtain barcoded nucleic acid fragments,

[0177] c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification status of the nucleic acid molecules in the sample based on the composition of the barcoded nucleic acid fragments.

[0178] 2. A method according to embodiment 1, wherein the sample comprises at least one cell.

[0179] 3. A method according to any one of embodiments 1-2, wherein the sample is a single cell.

[0180] 4. The method according to any one of embodiments 1-3, wherein the nucleic acid molecules in the sample are one or more DNAs in a single cell.

[0181] 5. The method according to any one of embodiments 2-4, wherein the cells are selected from the group consisting of cells isolated from an organism, cells cultured in vitro, primary cells, and discrete cells from explants.

[0182] 6. A method according to any one of embodiments 2-5, wherein the cells are fixed.

[0183] 7. A method according to any one of embodiments 1-6, which includes separating the sample into single cells.

[0184] 8. A method according to any one of embodiments 1-7, wherein the sample includes at least one organelle.

[0185] 9. A method according to embodiment 8, wherein the organelles include a nucleus, chloroplasts and / or mitochondria.

[0186] 10. A method according to any one of embodiments 1-9, wherein the sample is a single cell nucleus.

[0187] 11. A method according to any one of embodiments 1-10, wherein the nucleic acid molecules in the sample are one or more DNAs in a single cell nucleus.

[0188] 12. The method according to any one of embodiments 9-11, wherein the cell nucleus is derived from a cell selected from the group consisting of a cell isolated from an organism, a cell cultured in vitro, a primary cell, and a discrete cell from an explant.

[0189] 13. A method according to any one of embodiments 1-12, which comprises separating the sample into single cell nuclei.

[0190] 14. The method of any one of embodiments 1-13, comprising exposing the nucleic acid molecule from the sample.

[0191] 15. The method of embodiment 14, wherein the exposing comprises using a protease.

[0192] 16. The method according to any one of embodiments 1-15, comprising distributing the at least one nucleic acid molecule, the endonuclease, and the barcoded probe into discrete partitions.

[0193] 17. The method of 16, wherein the discrete partitions are wells or droplets.

[0194] 18. A method according to any one of embodiments 1-17, wherein the discrete partitions also include a ligase.

[0195] 19. The method of any one of embodiments 1-18, comprising providing a solid support having at least one barcoded probe attached thereto.

[0196] 20. The method of embodiment 19, wherein the solid support is beads.

[0197] 21. A method according to embodiment 20, wherein the beads are magnetic beads.

[0198] 22. The method according to any one of embodiments 1-21, wherein the barcoded probes comprise a barcode sequence, and the barcode sequences comprised by the barcoded probes attached to the same solid support are the same.

[0199] 23. A method according to embodiment 22, wherein the barcode sequence comprises at least 2 cellular barcode segments separated by a linker sequence.

[0200] 24. The method of any one of embodiments 1-23, wherein the barcoded probe comprises a universal priming sequence.

[0201] 25. The method of any one of embodiments 1-24, wherein the barcoded probe comprises a nucleic acid binding molecule.

[0202] 26. A method according to any one of embodiments 1-25, wherein the barcoded probe further comprises a unique molecular identification region.

[0203] 27. A method according to any one of embodiments 1-26, wherein the nuclease comprises a Class I restriction endonuclease, a Class II restriction endonuclease, a Class III restriction endonuclease, and a Class IV restriction endonuclease.

[0204] 28. The method of any one of embodiments 1-27, wherein the endonuclease comprises a modification-dependent endonuclease.

[0205] 29. The method of any one of embodiments 1-28, wherein the endonuclease comprises two or more endonucleases.

[0206] 30. The method of any one of embodiments 1-29, wherein the two or more endonucleases recognize or cleave different nucleic acid sites.

[0207] 31. A method according to any one of embodiments 1-30, wherein the nuclease is selected from any one or more of the following group: MspJI, AbaSI, BamHI, ClaI, EooRI, HindIII, HindII, KpnI, NotI, PstI, SalI, Sau3AI, SfiI, SmaI, XbaI and XhoI.

[0208] 32. The method according to any one of embodiments 1-31, comprising pre-treating the nucleic acid molecule before step a).

[0209] 33. According to the method according to embodiment 32, the pretreatment includes treating the nucleic acid molecule into a form that can be recognized and cut by a nuclease.

[0210] 34. The method of any one of embodiments 32-33, wherein the pretreatment comprises the use of a glycosyltransferase.

[0211] 35. The method of any one of embodiments 1-34, comprising obtaining the barcoded nucleic acid fragments from discrete partitions.

[0212] 36. The method of any one of embodiments 1-35, comprising amplifying the barcoded nucleic acid fragments.

[0213] 37. The method of any one of embodiments 1-36, comprising sequencing the barcoded nucleic acid fragments to determine the composition of the barcoded nucleic acid fragments.

[0214] 38. The method according to embodiment 37 comprises determining the sequence and / or modification state of the nucleic acid molecule by the number of barcoded nucleic acid fragments after determining the composition of the barcoded probe.

[0215] 39. A kit comprising the endonuclease of any one of embodiments 1-38, and the barcoded probe of any one of embodiments 1-38.

[0216] 40. An apparatus for performing the method of any one of embodiments 1-38.

[0217] Without intending to be bound by any theory, the following examples are merely intended to illustrate the analytical detection methods, preparation methods, kits, devices and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0218] Example

[0219] Example 1 5mC methylation sequencing

[0220] Cell microsphere preparation

[0221] The cell sample or cell nucleus sample suspension is mixed with acrylamide: dimethyl acrylamide solution to the target cell concentration, and polymerization inducers APS and TEMED are added to form droplets on the microfluidic chip. The cell encapsulation rate in the droplets is between 5-20%, wherein the concentrations are: 6% acrylamide, 0.2% dimethyl acrylamide, 0.2% APS, 0.2% TEMED. The droplet emulsion is allowed to stand at 55°C for 1 hour to polymerize. Demulsify with perfluorooctanol or other reagents and recover the cell microspheres. Wash with 1xPBS buffer to obtain Figure 2Subsequently, the microspheres were treated with 0.5% proteinase K in 1% SDS + 10 mM Tris buffer and incubated at 55°C for 1 hour to digest the proteins in the cells and leave pure genomic DNA. The protein digestion solution was washed away and the microspheres were stained with DAPI and counted.

[0222] Preparation of barcoding microspheres

[0223] Mix the primer AC-PCR handle-read1 primer modified with acrydite group and acrylamide: dimethyl acrylamide solution to the corresponding concentration, and add polymerization inducing agents APS and TEMED to form droplets on the microfluidic chip, where the concentrations are: 10mMAC-PCR handle-read1 primer, 6% acrylamide, 0.2% dimethyl acrylamide, 0.2% APS, 0.2% TEMED. The droplet emulsion is allowed to stand at 55°C for 1 hour to polymerize. Demulsify with perfluorooctanol or other reagents, recover the primer microspheres, and wash with 1xPBS buffer. Obtain primer microspheres: microsphere-PCR adaptor.

[0224] Barcode preparation:

[0225] Use the first extension primer PCR adaptor-barcode1 sequence-fix1 sequence to hybridize with the microsphere, use Klenow enzyme to perform extension reaction, and extend the primers on the microsphere. The first round of extension primers has a total of 96 (other numbers are also possible):

[0226] Microsphere-PCR adaptor

[0227] PCR adaptor-barcode1 sequence-fix1 sequence

[0228] The reaction system is as follows: 1x Klenow Buffer, 10mM dNTP, 1μ / μl Klenow Enzyme, 10mM extension primer barcode1-96, 10μl primer microspheres, a total of 25ul system. Add one extension primer to each well of the 96-well plate.

[0229] The primer microspheres were denatured at 95° C. and washed, and finally primer microspheres with the following structure were obtained: microsphere-PCRadaptor1-fix1 sequence.

[0230] Then it is hybridized with the second extension primer fix1-barcode2 sequence-fix2 sequence, and the extension reaction is carried out with Klenow enzyme. The second round of extension primers has a total of 96, and other numbers are also possible:

[0231] Microsphere-PCR adaptor-barcode1-fix1

[0232] fix1-barcode2 sequence-fix2 sequence

[0233] The reaction system was the same as that of the first round of extension.

[0234] The primer microspheres were denatured at 95° C. and washed, and finally primer microspheres with the following structure were obtained: microspheres-PCRadaptor1-fix1-barcode2-fix2.

[0235] Then it is hybridized with the third extension primer fix1-barcode3 sequence-capture sequence, and the extension reaction is carried out with Klenow enzyme. The third round of extension primers has a total of 96, and other numbers are also possible:

[0236] Microsphere-PCR adaptor1-fix1-barcode2-fix2

[0237] fix2-barcode3 sequence-capture sequence

[0238] The reaction system was the same as that of the first round of extension.

[0239] The primer microspheres are denatured, washed, and then hybridized with the fourth primer to form the final experimental microspheres, whose molecular structure is as follows:

[0240]

[0241] On board

[0242] Using a microfluidic chip, cell microspheres, barcoding microspheres, and molecular biology enzyme reaction solutions were injected and encapsulated into oil-in-water droplets. The reaction system was: molecular biology reagents, 1x T4 DNA ligase buffer, 1μ / μl T4 DNA ligase, and 1μ / μl MspjI. After the droplets were formed, they were reacted at 37°C for 1 hour. Figure 3 The droplets shown contain two microspheres in each droplet, namely, a cell microsphere and a barcode microsphere.

[0243] Building a database

[0244] Perfluorooctanol was added to the above emulsion to break the emulsion, remove the oil, and 0.8x volume of XP magnetic beads were used to purify the DNA in the aqueous phase. In vitro reverse transcription was performed using the NEB IVT kit to obtain aRNA. The reaction system for in vitro reverse transcription was: DNA, 10x IVT Buffer, 2μl IVT enzyme, and the reaction was carried out at 37°C for 3 hours.

[0245] The aRNA product was purified using RNA XP magnetic beads, and after quantification, the aRNA was reverse transcribed using Illumina read2-N6 primer. The reverse transcription system was: 1ug aRNA, 10uM RT primer, 10x RT buffer, 1ul RT enzyme, and reacted at 42°C for 1 hour. The obtained cDNA product was amplified using Illumina P5 and P7 primers to obtain a sequencing library. The DNA methylation status of the sample was analyzed using single-cell sequencing technology.

[0246] Test results

[0247] Human 293T and mouse 3T3 cell lines were mixed in a 3:1 ratio and high-throughput experiments were performed according to the above steps. A total of 1002 cells were identified after bioinformatics analysis. By comparing the genomic origins, 719 human-derived cells, 243 mouse-derived cells, and 40 mixed-origin cells were determined. The results are as follows Figure 4 As shown, this method is capable of detecting the omics information of single cells at a high throughput.

[0248] Three human cell lines, 293, Jurkat, and Hela, were mixed for high-throughput experiments, and a total of 2743 cells were obtained. The average methylation sites per cell were 53919. After clustering, the methylation sites could be divided into three groups: Figure 5 ), and cell line bulk methylation data ( Figure 6 ) to compare the correlations, we can identify which cell types the three types of cells correspond to. The results are as follows Figure 5-Figure 7 As shown, this method can effectively identify methylation information in single cells and has a good correlation with bulk methylation information ( Figure 7 ).

[0249] Using complex sample experiments, we obtained various cell numbers and the distribution of cell methylation sites. Using bone marrow mononuclear cell samples (BMMC), we conducted high-throughput experiments and obtained a total of 3533 cells, with an average of 2207 methylation sites per cell. After cell classification annotation, we obtained the following 17 cell groups: Figure 8 It is shown that the method of the present application can be used to analyze single-cell methylation information of multiple cells in complex samples.

[0250] Example 2 5hmC sequencing

[0251] The cell sample was pre-treated with galactosylation by galactosylation, and the 5hmC base site in the genome was galactosyl modified, and then the cell sample was prepared into a cell microsphere according to the method of Example 1. The cell microsphere and the primer microsphere were encapsulated into droplets, and the AbaSI enzyme and DNA ligase were simultaneously introduced into the droplets. After the AbaSI enzyme cuts the downstream of the 5hmC site glycosylated by galactosyltransferase to form a gap, it is connected to the adapter on the primer microsphere under the action of DNA ligase, thereby realizing the detection of the 5hmC site of a single cell.

[0252] Example 3 Genome Sequencing

[0253] Prepare cell microspheres according to the method of Example 1, and then use different sequence-specific restriction endonucleases to cut into multiple DNA fragments, and then connect with the adapter on the primer microsphere, so as to achieve the detection of single cell genome. In order to ensure that the genome is evenly cut into small fragments, enzymes such as MspI, HpaII, DpnI or HpyCH4IV with 4 base recognition sequences, and EcoRI, BamI or XbaI with 6 base recognition sequences can be used. For example, in one example, the cell microspheres and the primer microspheres with two adapter ends, which can be matched and connected with MspI and EcoRI restriction gaps, are also wrapped in droplets, and MspI, EcoRI and DNA ligase are introduced at the same time. The genomic DNA is cut into small fragments of 200-800bp under the action of MspI and EcoRI, and connected with the adapter with the same barcode sequence, but matching MspI and EcoRI restriction gaps, so as to achieve uniform detection of single cell genome.

Claims

1. A method for analyzing nucleic acid molecules in a sample, the method comprising: a) contacting the nucleic acid molecules in the sample with an endonuclease to obtain one or more nucleic acid fragments; b) contacting the one or more nucleic acid fragments with at least one barcoding probe to obtain barcoded nucleic acid fragments, c) determining the composition of the barcoded nucleic acid fragments, and determining the sequence and / or modification status of the nucleic acid molecules in the sample based on the composition of the barcoded nucleic acid fragments. The method according to claim 1 , wherein the sample comprises at least one cell.

3. The method according to claim 1, wherein the nucleic acid molecules in the sample are one or more DNA. The method of claim 1 , comprising separating the sample into single cells. The method according to claim 1 , wherein the sample is a single cell nucleus.

6. The method according to claim 1, wherein the nucleic acid molecules in the sample are one or more DNA.

7. The method according to claim 1, comprising exposing the nucleic acid molecule from the sample, preferably using a protease.

8. The method of claim 1, comprising partitioning the at least one nucleic acid molecule, the endonuclease, and the barcoded probe into discrete partitions.

9. The method of claim 8, wherein the discrete partitions are wells or droplets.

10. The method according to claim 8, wherein the discrete partitions further comprise a ligase, preferably T4 ligase.

11. The method of claim 1, comprising providing a solid support, preferably a bead, to which is attached at least one barcoded probe. 12 . The method of claim 1 , wherein the barcoded probes comprise a barcode sequence, and the barcode sequences comprised by the barcoded probes attached to the same solid support are identical.

13. The method of claim 12, wherein the barcode sequence comprises at least 2 cellular barcode segments separated by a linker sequence.

14. The method of claim 1, wherein the barcoded probe comprises one or more selected from the group consisting of a universal priming sequence, a nucleic acid binding molecule, and a unique molecular identification region.

15. The method of claim 1, wherein the endonuclease comprises a modification-dependent endonuclease, preferably a methylation-sensitive endonuclease.

16. The method according to claim 1, wherein the endonuclease is selected from any one or more of the following groups: MspJI, AbaSI, BamHI, ClaI, EooRI, HindIII, HindII, KpnI, NotI, PstI, SalI, Sau3AI, SfiI, SmaI, XbaI and XhoI.

17. The method according to claim 1, comprising, before step a), pre-treating the nucleic acid molecule, wherein the pre-treating comprises treating the nucleic acid molecule into a form capable of being recognized and cleaved by an endonuclease.

18. The method of claim 1, comprising sequencing the barcoded nucleic acid fragments to thereby determine the composition of the barcoded nucleic acid fragments.

19. A kit comprising the endonuclease according to any one of claims 1 to 18, and the barcoded probe according to any one of claims 1 to 18.

20. Apparatus for carrying out the method of any one of claims 1-18.

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