阅读说明：本技术 用于检测细胞内一个或多个dna分子内相互作用的方案 (Protocol for detecting one or more DNA intramolecular interactions in a cell ) 是由 戴晓光 M·彭德尔顿 于 2020-05-22 设计创作，主要内容包括：一种用于检测细胞内一个或多个DNA分子内的元件之间的相互作用的方法,其中元件在一级DNA序列中不相邻,该方法包括：a)提供细胞,其中紧邻的一个或多个DNA分子内的元件是交联的；b)同时裂解细胞和机械地分裂细胞内的DNA分子；c)邻位连接一个或多个分裂DNA分子；d)逆转连接DNA分子中的交联；e)对连接DNA分子进行测序；以及f)分析测序数据以检测细胞内一个或多个DNA分子内元件之间的相互作用。(A method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in primary DNA sequence, the method comprising: a) providing a cell in which elements within one or more DNA molecules in close proximity are cross-linked; b) simultaneously lysing the cells and mechanically dividing the DNA molecules within the cells; c) (ii) vicinal ligation of one or more split DNA molecules; d) reversing the cross-linking in the ligated DNA molecules; e) sequencing the ligated DNA molecules; and f) analyzing the sequencing data to detect interactions between one or more DNA intramolecular elements within the cell.)

1. A method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in primary DNA sequence, the method comprising:

a) providing a cell in which elements within one or more DNA molecules in close proximity are cross-linked;

b) simultaneously lysing said cells and mechanically dividing said DNA molecules within said cells;

c) (ii) ligating the one or more split DNA molecules in proximity;

d) reversing the cross-linking in the ligated DNA molecules;

e) sequencing the ligated DNA molecules; and

f) analyzing the sequencing data to detect interactions between the one or more DNA intramolecular elements within the cell.

2. The method of claim 1, wherein the cells are lysed and the DNA molecules are split by bead milling.

3. A method for detecting interactions between elements within one or more DNA molecules within a cell or nucleus, wherein the elements are not adjacent in primary DNA sequence, the method comprising:

a) providing a cell or nucleus in which elements within one or more DNA molecules in close proximity are cross-linked;

b) mechanically disrupting the DNA molecule within the cell by bead milling;

c) (ii) ligating the one or more split DNA molecules in proximity;

d) reversing the cross-linking in the ligated DNA molecules;

e) sequencing the ligated DNA molecules; and

f) analyzing the sequencing data to detect interactions between the one or more DNA intramolecular elements within the cell.

4. The method according to any one of claims 1 to 3, further comprising an initial step of cross-linking the DNA molecules and/or DNA interacting proteins.

5. The method of claim 4, wherein cross-linking is achieved by treating the cells with formaldehyde and/or disuccinimidyl glutarate (DSG).

6. The method of any one of the preceding claims, further comprising the step of blunt-terminating the DNA fragments produced in step (b) prior to step (c).

7. The method of any one of the preceding claims, further comprising the step of adding a linker to the ends of the DNA fragments after step (c) or (d).

8. The method of claim 7, wherein the linker is a sequencing linker.

9. The method of claim 8, wherein the sequencing adaptor is a PCR sequencing adaptor.

10. The method of any one of the preceding claims, further comprising a step of purifying the DNA fragment after step (d).

11. The method of any one of the preceding claims, further comprising selecting a DNA fragment of a desired size after step (b), (c) or (d).

12. The method of any one of the preceding claims, further comprising the step of enriching for one or more DNA molecules of interest prior to step (e).

13. The method of claim 12, wherein the DNA fragments of interest are enriched by hybridizing one or more labeled oligonucleotides to one or more regions of interest within the DNA molecule and selecting labeled DNA molecules.

14. The method of claim 13, wherein the oligonucleotide is labeled with an affinity tag and the labeled DNA molecule is selected by binding to a binding partner of the affinity tag.

15. The method of any one of the preceding claims, wherein sequencing is performed by a nanopore-based method.

16. The method of any one of the preceding claims, wherein the DNA molecule comprises a chromosomal sequence and/or an extrachromosomal sequence.

17. The method of any one of the preceding claims, wherein the step of analyzing the sequencing data comprises identifying tandem sequences from different elements within the one or more DNA molecules, thereby detecting an interacting element in one or more DNA elements.

18. The method of any one of the preceding claims, wherein the element is a locus within a chromosome.

Technical Field

The present invention relates generally to a method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in the primary DNA sequence.

Background

What is needed is a technology that can provide information that can improve understanding of the spatial organization of the genome. The slow developing prior art includes steps that introduce bias. These deviations prevent the resolution of DNA interactions in the entire genomic sequence.

Disclosure of Invention

The present inventors have identified novel methods for detecting and analyzing interactions between elements within one or more DNA molecules within a cell. In particular, these methods enable the detection of interaction elements that are not adjacent in the primary DNA sequence. The interaction information may provide an understanding of the conformational characteristics of the hierarchical organization of the support genome. In particular, these conformational features include the entire chromosomal domain, large-scale active and inhibitory compartments, topologically-associated domains, lamin-associated domains, nucleolar-associated domains, and individual circulatory interactions between one or more elements within the same or different chromosomes. These methods can also provide the same information when applied to heterogeneous metagenomic samples, in particular to identify interactions between bacterial chromosomes and their plasmids.

A key advantage of these methods is that the simultaneous steps of mechanical cell division and mechanical cross-linking DNA division mean that: because the number of steps in the scheme is small, the ability to cause errors is reduced. The single step cell lysis and DNA division makes these methods simplified and streamlined. Furthermore, importantly, these methods provide sequencing-based element interaction information that is sequence independent and thus not limited by the effects of enzyme targeting and like bias; these methods are not affected by chemical modifications of the DNA bases or by the accessibility of the DNA sequences. In particular, similar methods in the art utilize restriction enzymes in the cleavage step. This approach is not limited to the effects of genomic regions where the concentration of enzyme motifs is too low or too high. In contrast, mechanical fragmentation of DNA according to the present method allows for maximum mapping of sequencing data without sacrificing resolution.

Accordingly, provided herein is a method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in the primary sequence of DNA, the method comprising: a) providing a cell in which DNA molecules in close proximity to elements within one or more DNA molecules are cross-linked; b) simultaneously lysing the cells and mechanically lysing the DNA molecules within the cells; c) contiguously ligating one or more fragmented DNA molecules; d) reversing the cross-linking in the ligated DNA molecules; e) sequencing the ligated DNA molecules; f) the sequencing data is analyzed to detect interactions between elements within one or more DNA molecules within the cell.

Also provided is a method for detecting interactions between elements within one or more DNA molecules within a cell or nucleus, wherein the elements are not adjacent in the primary sequence of DNA, the method comprising: a) providing a cell or nucleus in which an element or a plurality of adjacent DNA molecules within an element are cross-linked; b) mechanical breakage of intracellular DNA molecules by bead beating; c) contiguously ligating one or more fragmented DNA molecules; d) reversing the cross-linking in the ligated DNA molecules; e) sequencing the ligated DNA molecules; f) the sequencing data is analyzed to detect interactions between elements within one or more DNA molecules within the cell.

Drawings

It is to be understood that the drawings are for purposes of illustrating particular embodiments of the invention and are not intended to be limiting.

Figure 1 illustrates an example of how the methods of the present disclosure may be used to provide information about interactions between elements within one or more DNA molecules. This example shows a heterogeneous mixture of intact cells in a sample tube. Then, a cross-linking agent is applied to the intact cells to cross-link the molecules within the cells. In particular, the cross-linking agent can cross-link a DNA molecule to an interacting DNA molecule, a DNA molecule to an interacting protein, and/or a protein to an interacting protein. The circular dotted line represents a cell, nucleus or any other vesicle. The lines labeled "genome a", "plasmid a" and "genome B" represent DNA molecules. The smaller overlapping gray circles represent proteins that interact with each other and simultaneously with the DNA molecule. Thus, the proteins in this schematic "bridge" one or more interacting elements within the DNA molecule. These protein-protein and protein-DNA interactions are cross-linked due to the use of cross-linking agents. Cells and DNA molecules are divided by a mechanical process of "bead milling". The split ends of the cross-linked DNA molecules are then ligated to split ends that are in close proximity to each other. In the top panel of the exemplary figure, the split genomic a DNA molecule is ligated to the split plasmid ADNA molecule in the case of individual intramolecular elements, wherein the individual intramolecular elements, thus meaning that the fragment ends of the individual cross-linked DNA molecules next to each other, are ligated. This example shows that the cross-linking can then be reversed and the ligated DNA molecule can be purified. In this example, the purified ligated DNA molecules from the top panel represent the tandem sequence of genomic a and plasmid a sequences, indicating that: the elements within these sequences interact with each other in the original cell from which they were derived. This example further shows that the purified DNA molecules can then be size selected and amplified by Polymerase Chain Reaction (PCR), followed by sequencing library preparation protocols (e.g., incorporation of one or more linkers, leader sequences, and/or hairpin loops) and sequencing.

Fig. 2 shows an exemplary bioinformatics analysis workflow whereby sequence reads are obtained by the method described in the embodiment of fig. 1, wherein the sequencing step to obtain the sequence reads is performed by a nanopore-based method. In FIG. 2, these sequencing reads are referred to as "nanopore MetaPore-C reads". Sequencing reads exemplified by MetaPore-C reads (tandem sequences) were locally aligned to the reference genomic sequence. As can be seen from the bottom left of fig. 2, regions of a single sequencing read may align with the same sequence present in more than one species/genome. Thus, the alignment path through each individual MetaPore-C sequence read was optimized to resolve the most likely species to which the sequencing reads aligned. This example further shows that genomic sequences can be isolated into "bins" of appropriate length (in bp) and aligned MetaPore-C sequencing reads can be assigned to the bins. The number of reads assigned to the bin can then be used to tabulate a contact map (heatmap) based on the frequency of the assigned reads that are adjacent to each other in the MetaPore-C sequencing reads.

Figure 3 shows data derived from exemplary methods showing the identification of intrachromosomal and extrachromosomal contacts (interactions) in probiotic samples. A shows a table of 15 known bacterial strains contained within an initial probiotic sample on which the method depicted in figure 1 was performed to determine the interactions between one or more DNA intramolecular elements within the cell. B and C show contact maps for each of the 15 bacterial strains representing a prepared according to the bioinformatics workflow set forth in fig. 2. D shows an average nucleotide density heatmap representing the degree of genomic similarity between the 15 bacterial strains of a (and their associated plasmids). E and F show bar graphs representing the number of contacts and the type of contacts per bacterial DNA molecule.

Detailed Description

It is understood that different applications of the disclosed products and methods may be tailored to specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes two or more polynucleotides, reference to "a transmembrane pore" includes two or more pores, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Method

There is provided a method for detecting interactions between elements within one or more DNA molecules within a cell, wherein the elements are not adjacent in a primary DNA sequence, the method comprising: a) providing a cell in which elements within one or more DNA molecules are cross-linked in close proximity; b) simultaneously lysing the cells and mechanically lysing the DNA molecules within the cells; c) contiguously ligating one or more fragmented DNA molecules; d) reversing the cross-linking in the ligated DNA molecules; e) sequencing the ligated DNA molecules; f) the sequencing data is analyzed to detect interactions between elements within one or more DNA molecules within the cell.

Also provided is a method for detecting interactions between elements within one or more DNA molecules within a cell or nucleus, wherein the elements are not adjacent in the primary sequence of DNA, the method comprising: a) providing a cell or nucleus in which an element or a plurality of adjacent DNA molecules within an element are cross-linked; b) mechanical breakage of intracellular DNA molecules by bead beating; c) contiguously ligating one or more fragmented DNA molecules; d) reversing the cross-linking in the ligated DNA molecules; e) sequencing the ligated DNA molecules; f) the sequencing data is analyzed to detect interactions between elements within one or more DNA molecules within the cell.

The method can be used, for example, to obtain information relating to the spatial organization of one or more DNA molecules (e.g., genomes) in a cell. In particular, the method can provide information about the hierarchical organization of the genome in the cell. Exemplary conformational features that support genomic hierarchal organization that can be resolved by the present methods include the entire chromosomal domain, large-scale active and inhibitory compartments, topologically-related domains, lamin-related domains, nucleolar-related domains, and individual circulatory interactions between one or more elements within the same or different chromosomes. These methods can also provide the same information when applied to heterogeneous metagenomic samples, in particular to identify interactions between bacterial chromosomes and their plasmids.

Interaction between elements

In any of the methods described herein, an "element" can refer to a portion of a nucleotide sequence of any size within one or more nucleic acid molecules. The nucleic acid molecule may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleic acid molecule may comprise one RNA strand hybridized to one DNA strand.

The nucleic acid molecule is preferably DNA, RNA or a DNA or RNA hybrid, most preferably DNA. The nucleic acid molecule may be double stranded. The nucleic acid molecule may be genomic DNA. The nucleic acid molecule may comprise single stranded regions and regions having other structures, such as hairpin loops, triplexes, and/or quadruplexes. A DNA/RNA hybrid may comprise DNA and RNA on the same strand. Preferably, the DNA/RNA hybrid comprises one DNA strand hybridized to an RNA strand.

The nucleic acid molecule can be of any length. For example, the nucleic acid molecule can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The target nucleic acid molecule can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100000 or more nucleotides or nucleotide pairs in length. The nucleic acid molecule may be the entire genome. The nucleic acid molecule may be the whole of all nucleic acid molecules contained within the cell. The nucleic acid molecule may be a sub-selection of all nucleic acid molecules contained within the cell. The nucleic acid molecule may be the entire DNA contained within the cell. The nucleic acid molecule may be a sub-selection of DNA contained within the cell, such as a single chromosome.

An element can be a portion of a nucleotide sequence of any size within one or more nucleic acid molecules. An element may be a locus defined by specific coordinates assembled according to a given genomic reference. Thus, an element can be one or more loci within a chromosome. An element can be a coding or non-coding sequence of a genome. The elements may be nucleotide sequences within heterochromatin (closed chromatin) or euchromatin (open chromatin). The element may be a cis regulatory element or a cis regulatory module. The elements may be promoters, enhancers, silencers, exons, introns. The element may be a binding site for a protein, such as a histone, a transcription factor, and/or a trans-acting factor. The element may be part of an open chromatin flanking histones. The elements may be CpG islands. The element may be a genetic desert. The element may be a transcription factor binding motif. An element may be a region comprising small nucleotide polymorphisms in linkage disequilibrium with one another. The element may be a single SNP, CpG, or a single nucleotide base. In any of the methods described herein, the elements are not adjacent in the primary nucleotide sequence of one or more DNA molecules.

In any of the methods described herein, "interaction" may refer to any form of direct or indirect contact between elements within one or more DNA molecules. The element may be comprised within one or more DNA molecules within one or more cells. Interaction may refer to indirect or direct interaction between elements, where the elements are not contiguous in the primary DNA sequence. Thus, the interaction may provide an indication of the 3D genomic structure. In particular, the interaction may further provide an indication of an accurate map of the particular organization of elements within the DNA molecule. The interaction may be two or more elements in proximity to each other. Thus, in the context of describing the spatial organization of elements within a DNA molecule, elements that are in proximity to each other may also be considered to interact, regardless of whether the proximity of two or more elements produces any functional consequence. As used herein, the term "proximate" refers to the distance between two elements in three-dimensional space. For example, DNA sequence elements in chromosomes that are close in primary sequence (e.g., within about 10, 50, 100, 150, 200, or 250bp or more) are always in close proximity to each other. In some cases, DNA sequence elements that are further apart in the primary sequence in a chromosome (e.g., more than about 200; 250; 300; 400; 500; 1000; 1500; 2000; 5000; 10,000; 25,000; 50,000; 100,000; 250,000; 500,000; or 1,000,000bp apart) may be in close proximity to each other because of the tertiary or quaternary structure of the chromosome. In some cases, DNA sequence elements located on different chromosomes can be in close proximity to each other because of the quaternary structure of the chromosome. In some cases, a nucleic acid sequence element is distal with respect to a primary sequence in that one or more elements are chromosomal DNA sequence elements and one or more other elements are RNA (or cDNA) sequence elements. Thus, nucleic acid sequence elements may be or may be within different nucleic acid molecules. In this case, two or more nucleic acid sequence elements may be in close proximity to each other as they form a complex. For example, a non-coding RNA can be associated with one or more DNA sequence elements in a genome. Thus, in any of the methods described herein, DNA sequence elements may be considered to interact because of their proximity.

Two or more elements may interact simultaneously. The elements may interact directly or may interact indirectly. Indirect interactions may be mediated by direct interactions with one or more proteins. For example, an indirect interaction may be represented by a protein complex that binds both enhancer and promoter, where the two elements are more than 100,000bp apart from each other in terms of primary sequence.

Sample (I)

In any of the methods described herein, the sample can be any suitable sample. The sample should contain one or more DNA molecules. The sample is typically a sample known to contain or suspected of containing one or more DNA molecules. The sample may contain one or more cells.

The sample may be a biological sample. The disclosed methods can be performed in vitro on a sample comprising cells from any organism or microorganism. The organism or microorganism is typically an archaebacterium, a prokaryote or a eukaryote, and typically belongs to one of five kingdoms: the kingdom Plantae, the kingdom Animalia, the kingdom fungi, the kingdom Prokaryotae, and the kingdom Protista. The method may be performed in vitro on a sample obtained or extracted from any virus.

The sample is preferably fluid-based. The sample typically comprises a bodily fluid. The body fluid may be obtained from a human or an animal. The human or animal may have, be suspected of having, or be at risk of having a disease. The sample may be urine, lymph, saliva, mucus, semen or amniotic fluid, but is preferably whole blood, plasma or serum. Typically, the sample is of human origin, but alternatively it may be from other mammals, such as from commercially farmed animals, such as horses, cattle, sheep or pigs, or alternatively may be pets, such as cats or dogs.

Alternatively, samples of plant origin are typically obtained from the following commercial crops: such as cereal crops, legume crops, fruits or vegetables, for example wheat, barley, oats, oilseed rape, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton, tea or coffee.

The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater or river water, and reagents for laboratory testing. For example, the sample can be an environmental sample comprising a heterogeneous mixture of cells from two or more different organisms.

The sample may be processed prior to application to the methods described herein, for example, by centrifugation or through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately after acquisition. It is also generally possible to store the sample prior to analysis, preferably below-70 ℃.

The sample preferably comprises genomic DNA. The sample may be one or more nuclei.

Cross-linking

In any of the methods described herein, a sample is provided in which elements within one or more immediately adjacent DNA molecules are cross-linked. The sample may comprise one or more cells. The cells may be crosslinked by any crosslinking agent suitable for use in the methods described herein. By applying the cross-linking agent to the cells, a "snapshot" of the spatial organization of the DNA molecules can be obtained, thereby obtaining the interaction between one or more elements within the DNA molecule within the cell. One or more cross-linking agents may be applied to cells within the sample to covalently bond molecules that are adjacent to each other. Preferably, the one or more cross-linking agents cross-link DNA to DNA, DNA to protein, and protein to protein. More preferably, the method may further comprise cross-linking the DNA molecules and/or DNA interacting proteins.

Preferably, the cross-linking agent in the methods described herein reacts with amino groups in the protein and/or imino and amino groups in the DNA, thereby enabling cross-linking to be formed between any or all of these groups. Exemplary crosslinkers include formaldehyde, disuccinimidyl glutarate (DSG), bis [2- (N-succinimide-oxycarbonyloxy) ethyl ] sulfone (BSOCOES), disuccinimidyl Dibutyrate (DSBU), 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB), dimethyl adipate Dihydrochloride (DMA), dimethyl pimelate (DMP), dimethyl suberate (DMS), dithiobis (succinimidyl propionate) (DSP), disuccinimidyl suberate (DSS), disuccinimidyl sulphoxide (DSSO), disuccinimidyl tartrate (DST) Dimethyldithiodipropionate (DTBP), ethylene glycol bis (succinimidyl succinate) (EGS), sulfo-EGS, tris- (succinimidyl) aminotriacetate (TSAT). Preferably, crosslinking is achieved by treating one or more cells with a crosslinking agent. Even more preferably, crosslinking is achieved by treating the cells with formaldehyde and/or disuccinimidyl glutarate (DSG).

The conditions for the crosslinking step can be appropriately selected by those skilled in the art. For example, it is within the routine skill of the skilled artisan to select an appropriate buffer and/or temperature and then use any given crosslinking agent to achieve the desired degree of crosslinking.

Cleavage and cleavage

In any of the methods described herein, the cross-linked cells can be lysed. DNA molecules from cells can be split at the same time as or after cell lysis. In any of the methods described herein, the cross-linked cells can be lysed while the DNA molecules within the cells are being split.

The cells may be lysed by any suitable protocol. Cell lysis may be performed by physical disruption or solution-based protocols. The physical interruption scheme may be a "mechanical" scheme. Physical disruption and mechanical protocols can divide lysed cells while DNA molecules contained within the cells are being divided. An exemplary physical disruption scheme suitable for use in the disclosed method is the use of a waring blender polytron. Another exemplary physical disruption scheme suitable for use in the disclosed method is the use of a Dounce homogenizer. Another exemplary physical disruption scheme suitable for use in the disclosed method is the use of a Potter-Elevehjem homogenizer. Another exemplary physical disruption scheme suitable for use in the disclosed methods is the use of sonication. Another exemplary physical disruption protocol suitable for use in the disclosed methods is the use of freeze-thaw cycles. Another exemplary physical disruption protocol suitable for use in the disclosed methods is the use of a pestle and mortar. A preferred example of a physical disruption suitable for use in the disclosed method is bead milling.

Bead milling involves combining beads with a sample and physically agitating the combination, thereby causing the cells in the sample to divide. The beads used in the bead mill can be any suitable material suitable for use in the methods described herein. For example, the beads may be ceramic, metal or glass. Preferably, the beads are glass. The beads may be of any size suitable for use in a sample in a mechanical physical disruption protocol. The beads may be less than 5mm in diameter. The beads may be less than 3mm in diameter. The beads may be less than 1mm in diameter. Preferably, the beads have a diameter of between 0.1mm and 1 mm. Even more preferably, the beads are 0.5mm in diameter. In addition to the size and number of beads used, the combined sample and beads may be agitated by any suitable method depending on the volume of the sample. Agitation of the cells by bead milling can simultaneously lyse the cells and divide the DNA molecules contained within the cells.

One exemplary method of agitation is vortexing. Any standard laboratory bench top vortex can be used. Preferably, the vortex has a strength setting. Preferably, the agitation step is performed at the highest intensity selectable on a standard laboratory bench top vortex. Any of the bead milling steps in the methods described herein can be performed through a single agitation cycle. The stirring period may be less than 30 minutes. The stirring period may be less than 20 minutes. The stirring period may be 15 minutes.

Any of the bead milling steps in the methods described herein can be performed by a stirring and cooling cycle. For example, the bead milling step may include 5 agitation cycles separated by a cool incubation step. The agitation cycle may last for any suitable period of time. The separate cooling incubation step may last for any suitable period of time.

The sample subjected to bead milling is preferably kept cool during the bead milling protocol. The bead milling may be performed at sub-ambient temperatures. The bead milling may be performed at below 18 ℃. The bead milling may be performed at 4 ℃. The sample subjected to bead milling may be incubated at a temperature below room temperature between the stirring steps. The sample subjected to bead milling may be incubated at a temperature below 18 ℃ between the stirring steps. The samples subjected to bead milling can be incubated at 4 ℃ between the stirring steps. Preferably, the bead mill comprises three 3 minute vortexing steps, wherein vortexing is performed at the highest intensity selectable on standard laboratory bench-top vortexes, each vortexing being incubated at 4 ℃ or 2 minutes on ice.

The DNA molecules contained within the cells undergoing lysis can then be cleaved. Although the cross-linked cells can be lysed in any of the methods described herein, the DNA molecules within the cells are cleaved at the same time. The simultaneous mechanical cell division and mechanical cross-linking DNA division steps mean: because the number of steps in the scheme is small, the ability to cause errors is reduced. The single step cell lysis and DNA division makes these methods simplified and streamlined.

For example, the bead milling parameters described may be combined in any manner to achieve cell lysis and/or a desired degree of DNA division.

The DNA molecule may be cleaved by any suitable method. DNA cleavage involves breaking down a DNA molecule into smaller fragments. The DNA molecule may be from a lysed cell or nucleus. Preferably, the cells are lysed and the DNA molecules contained within the cells are simultaneously split in a single step.

The DNA can be mechanically cleaved. Preferably, the method comprises: lysing cells in which elements within one or more DNA molecules in close proximity are cross-linked, and simultaneously mechanically dividing the DNA molecules within the cells. Preferably, the cells are mechanically disrupted and the DNA molecules are mechanically disrupted. Even more preferably, the cell is mechanically disrupted and the DNA molecules contained within the cell are simultaneously mechanically disrupted in a single step. Any of the above mechanical methods of lysing cells can divide the DNA.

The methods of the present disclosure may include mechanical disruption by bead milling. Bead milling can be applied, for example, to intact cells, intact nuclei, lysed cells, lysed nuclei, and/or to isolate DNA. Preferably, the cross-linked cells of the methods described herein are lysed while the DNA molecules within the cells are disrupted. Longer bead milling durations or greater bead milling strengths result in the DNA molecules being broken into smaller fragments. The simultaneous mechanical cell division and mechanical cross-linking DNA division steps mean: because the number of steps in the scheme is small, the ability to cause errors is reduced.

The single step mechanical lysis of cells and mechanical DNA division allows these methods to be simplified and streamlined. Furthermore, the disclosed methods provide DNA fragments that have been obtained in a sequence independent manner. This means that the cleavage step of the method involving mechanical cleavage (e.g. bead milling) is not affected by too much or too little restriction enzyme motifs or chemical modification of the DNA. Thus, the absence of bias in the mechanical disruption step of the method can improve genome coverage when interactions between elements are detected. Mechanical fragmentation of DNA according to the present method allows for maximum mapping of sequencing data without sacrificing resolution. The fragment size can be fine-tuned by different aspects of the mechanical disruption step (e.g. bead milling).

The DNA molecule can be split into any size suitable for the sequencing platform chosen for application in the methods described herein. The mechanical cleavage step can result in a DNA molecule of at least about 100bp, e.g., at least about 250bp, at least about 500bp, at least about 1kbp, at least about 2kbp, at least about 5kbp, at least about 10kbp, or at least about 15 kbp. For example, a fragment may have a length of about 100bp to about 15kbp, e.g., about 250bp, about 500bp, about 1kbp, or about 2kbp up to about 5kbp, about 10kbp, or about 15 kbp.

Ortho-position ligation

In any of the methods described herein, the split DNA molecules can be ortho-linked. Proximity ligation in this context has the effect of forming concatemer sequences, whereby a DNA fragment representing an element within the original DNA molecule or molecules is covalently linked to other fragments that are adjacent in three-dimensional space but not in the primary sequence. Thus, concatemer sequences indicate which DNA elements interact with each other in one or more DNA molecules.

After the cleavage step, the cleaved DNA molecule sample is preferably diluted. Without dilution, spurious proximity ligation events with cross-linked cleaved DNA molecules that are randomly adjacent to other cross-linked cleaved DNA molecules in solution may result. As an alternative to diluting the sample solution, the DNA fragments may be separated on an agarose gel based on their size, thereby reducing the likelihood of spurious proximity ligation events with cross-linked split DNA molecules that are randomly adjacent to other cross-linked split DNA molecules.

The proximity ligation step in any of the methods described herein can be performed with any suitable DNA ligase known in the art. Exemplary ligases and kits may include T4 DNA ligase, Tfi DNA ligase, DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, small size DNA ligase, Blunt/TA premix of NEB or Rapid ligation of NEB^TMOne or more of the kits. Preferably, the ligase is capable of ligating blunt ends of double-stranded DNA fragments.

In any of the methods described herein, the proximity ligation is performed in order to provide a 5C or Hi-C library.

Before the ortho-ligation step but after the DNA molecule cleavage step, the still cross-linked DNA-cleaving DNA molecules may undergo "end repair". The DNA fragment end repair may facilitate ligation at the ortho position. Any suitable DNA end repair protocol may be used. An exemplary product for repairing cleaved DNA ends is NEBNext end repair enzyme. Preferably, the split DNA molecules have "blunt ends" such that DNA fragments with blunt ends are provided for proximity ligation. Any enzyme or kit capable of blunt-terminating a double-stranded DNA molecule can be used in the present method.

In any of the methods described herein, after the ortho-ligation step, the crosslinking may be reversed by any suitable method. The method of reversing crosslinking suitable for use in the methods described herein may depend on the crosslinking agent or agents used in the crosslinking step of the methods described herein. For example, where formaldehyde is the crosslinking agent used in the methods described herein, the crosslinking may be reversed by any of the following: incubated with high salt (e.g., NaCl) and incubated at 65 ℃ for a long period of time, or with Tris HCl buffer bound RNaseA and proteinase K at 65 ℃ for a long period of time.

The ligated DNA fragments may, for example, have a length of at least about 250bp, at least about 500bp, at least about 1kbp, at least about 2kbp, at least about 5kbp, at least about 10kbp, or at least about 15 kbp. For example, a fragment may have a length of about 250bp to about 100kbp, e.g., about 2kbp, about 5kbp, or about 10kbp up to about 15kbp, about 50kbp, or about 100 kbp.

DNA purification

In any of the methods described herein, the DNA molecule may be purified after the ligation and cross-linking reversal steps. In any of the methods described herein, the DNA may be performed by any method known in the art suitable for purifying DNA. Preferably, the purification method applied in the present method provides DNA that is sufficiently pure for sequencing. Exemplary methods for purifying DNA include organic extraction methods such as phenol-chloroform and ethanol precipitation, Chelex extraction purification, and solid phase purification, as well as any DNA purification kit known in the art. Preferably, the purification step used in the methods described herein uses Solid Phase Reversible Immobilization (SPRI) beads.

Size selection

Any of the methods described herein may include the step of selecting a DNA of a desired size at any suitable stage. Fragments of a desired size may be selected after cleaving the cross-linked DNA molecules and/or after ligating one or more of the cleaved DNA molecules in the ortho position and/or after reversing the cross-linking in the ligated DNA molecules. Size selection may be performed by any suitable method. In any of the methods described herein, any inclusion of a step comprising selecting a DNA fragment of a desired size will preferably occur immediately prior to any sequencing step. Exemplary DNA size selection methods include isolating DNA fragments on an agarose gel, followed by excision of the gel containing the DNA fragment of the desired size and purification, SPRI beads or BluePippin (Sage science).

The desired DNA fragment size may vary depending on what sequencing platform will be used to sequence the ligated DNA molecule. Fragment sizes between 200bp and 500bp are the first choice for Illumina-based sequencing methods. When the sequencing method from Oxford Nanopore Technologies is applied to the methods described herein, the required DNA fragment size is typically more than 500bp, preferably more than 1kb, even more preferably more than 3 kb.

For example, the sequencing platform of Oxford Nanopore Technologies can be used to sequence DNA molecules that are more than 3kb in length. Preferably, the ligated fragments that form the concatemer DNA fragments are of sufficient length to uniquely map to the reference genomic assembly. Even more preferably, the ligated fragments that form the concatemer DNA fragments are long enough and of high enough read quality to uniquely map to the reference genomic assembly.

Enrichment of

The methods disclosed herein may further comprise the step of enriching for one or more DNA molecules of interest. The DNA molecules of interest may be enriched at any stage in the process deemed appropriate. In some cases, the ligated DNA molecules may be enriched prior to sequencing. In other cases, the ligated DNA molecules may be enriched immediately prior to sequencing. In other cases, the ligated DNA molecules may be enriched after purification. In other cases, the ligated DNA molecules may be enriched after selection of DNA fragments of the desired size. In other cases, the ligated DNA molecules may be enriched after purification and size selection.

The DNA molecule of interest may be enriched by any suitable method. For example, a DNA molecule of interest may be a specific element whose interaction partner is of interest.

One exemplary enrichment method is by hybridizing one or more labeled oligonucleotides of complementary base sequences to one or more specific regions of interest within the DNA, wherein the label is an affinity tag, and wherein the DNA molecule of interest is isolated and thus enriched, by targeting the affinity tag with a binding partner for the affinity tag, and discarding any DNA not related to the binding partner. Exemplary affinity tags and binding partners are biotin and streptavidin.

Other exemplary enrichment methods are by reverse Polymerase Chain Reaction (PCR). Enrichment by inverse PCR in the context of the present method may comprise circularization of the ligated DNA molecule, and wherein a pair of primer sequences of base sequences complementary to a specific target region of the circularized DNA molecule (hence one or more elements of the DNA molecule) prime PCR extension in the inverse direction, and wherein the target region and its flanking (interacting) sequences are amplified.

Other exemplary enrichment methods are by semi-specific PCR. Enrichment by semi-specific PCR in the context of the present method may comprise treatment of the ligated DNA molecules with a mixture of end-preparing enzymes to generate ligatable ends of the dA-tails, wherein the ligatable ends may then be ligated to a universal PCR adaptor, and wherein sequence-specific primers for the target element of the DNA molecule may be combined with a single universal PCR primer comprised within the PCR adaptor and amplify the target and its flanking (interacting) sequences. Either side of the target can be studied with the corresponding primer design.

Joint

Any of the methods described herein can further comprise the step of adding a linker to the ends of the DNA fragments prior to sequencing the DNA. Preferably, the linker is a sequencing linker. The sequencing adaptor may be a PCR sequencing adaptor. Any suitable sequencing linker may be used in the methods described herein, depending on the sequencing platform used, and any suitable sequencing platform may be used in the methods described herein. More preferably, a linker compatible with the Oxford Nanopore Technologies' sequencing platform is used in the methods described herein.

The Oxford Nanopore sequencing linker may comprise at least one single-stranded polynucleotide or non-polynucleotide region. For example, Y-linkers for nanopore sequencing are known in the art. The Y adaptor generally comprises (a) a double stranded region and (b) a single stranded region or a region that is not complementary at the other end. If the Y-linker comprises a single-stranded region, it may be described as having an overhang. The presence of the non-complementary region in the Y-linker imparts a Y-shape to the linker, since the two strands do not normally hybridize to each other, unlike the double-stranded portion. The Y-linker may comprise one or more anchors.

The Y-linker preferably comprises a leader sequence which preferably spirals into the pore. The leader sequence typically comprises a polymer. The polymer is preferably negatively charged. The polymer is preferably a polynucleotide, such as DNA or RNA, a modified polynucleotide (e.g. abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. The leader preferably comprises a polynucleotide, and more preferably a single stranded polynucleotide. The single stranded leader sequence most preferably comprises a single strand of DNA, such as a poly dT segment. The leader sequence preferably comprises one or more spacers.

The leader sequence may be of any length, but is typically 10 to 150 nucleotides in length, for example 20 to 150 nucleotides. The length of the leader is generally dependent on the membrane-embedded nanopore used in the method.

The leader sequence preferentially screws into the transmembrane pore and thereby facilitates movement of the polynucleotide through the pore.

The Y-linker may comprise a capture sequence, an affinity tag or a pore-based strand, which is revealed upon unfolding of the double-stranded region to which the linker is attached. The capture sequence or tag serves to prevent diffusion of the second strand of the DNA molecule away from the nanopore as the DNA molecule unfolds with the first strand of the DNA molecule passing through the pore, wherein the pore is bound to a tether or labeled with an oligonucleotide comprising a sequence complementary to the capture sequence in the Y-linker, an affinity partner for the tag on the Y-linker. Any method known in the art can be used to attach the linker to the DNA molecule. Ligases (such as T4 DNA ligase, e.coli DNA ligase, Taq DNA ligase, Tma DNA ligase and 9 ° N DNA ligase) may be used to ligate one or both of the adaptors. Alternatively, linkers can be added to the DNA molecules using the methods discussed below.

In one embodiment, the method comprises modifying one or more molecules in the sample such that they comprise a Y-linker at one end and a hairpin loop at the other end. Any modification may be used.

Hairpin loop linkers for nanopore sequencing are known in the art. The hairpin loop may be provided at one end of the DNA molecule, the method preferably further comprising providing the DNA molecule with a hairpin loop at one end of the DNA molecule. The two strands of the DNA molecule may be joined at one end to a hairpin loop.

Sequencing

The methods described herein may further comprise the step of sequencing the ligated DNA molecules. The step of sequencing the ligated DNA molecules may be to determine all or part of their sequence. Any suitable sequencing technique may be employed to determine the sequence of the ligated DNA molecules. In the methods of the present disclosure, the ligated DNA molecules can be sequenced using high throughput (so-called "second generation", "third generation" and "next generation" techniques).

In the second generation of technology, a large number of DNA molecules are sequenced in parallel. Typically, tens of thousands of molecules are anchored at a high density at a given location and sequence is determined in a process that relies on DNA synthesis. The reaction typically consists of successive reagent delivery and washing steps (e.g., to allow incorporation of reversibly labeled terminator bases) and scanning steps (to determine the order of base incorporation). Array-based systems of this type are commercially available from, for example, Illumina, Inc.

Third generation techniques are generally defined as not requiring the sequencing process to be stopped between detection steps. For example, the base-specific release of hydrogen ions that occurs during incorporation can be detected in the context of a microwell system (e.g., Ion Torrent system available from Life Technologies). Similarly, in pyrosequencing, the base-specific release of pyrophosphate (PPi) is detected and analyzed. In nanopore sequencing techniques, a DNA molecule is passed through or positioned near a nanopore, and the identity of individual bases is determined after the DNA molecule is moved relative to the nanopore. For example, this type of system is commercially available from Oxford Nanopore Technologies. In another technique, the DNA polymerase is confined in a "zero mode waveguide" and the identity of the incorporated base is determined by fluorescent detection of gamma-labeled phosphonucleotides (see, e.g., Pacific Biosciences).

The methods described herein may include analyzing the assay data to detect interactions between one or more DNA intramolecular elements within the cell. Analyzing the sequencing data may include identifying tandem sequences from different elements within one or more DNA molecules, thereby detecting interacting elements in one or more DNA elements.

The following non-limiting examples illustrate the invention and are not intended to be limiting.

Example 1:

this example describes an exemplary laboratory workflow suitable for use in the present disclosure, a method for determining interactions between intracellular elements. In particular, the method is used to study interactions between genomic elements that are not adjacent in the primary sequence. In addition to simplifying assembly, the disclosed methods provide a way to associate a plasmid with its host genome when applied to a metagenomic sample. An alternative approach for determining interactions between one or more elements within a DNA molecule employs restriction enzyme cleavage of the cross-linked DNA prior to performing proximity ligation. However, restriction enzyme cleavage is rather time consuming and the choice of restriction enzyme is influenced by the nucleotide composition of the genome in the sample, which is not always known in advance, especially when performing metagenomic studies. The present disclosure provides a method to avoid restriction enzyme cleavage by using mechanical disruption (e.g., bead milling) to simultaneously lyse cells and divide DNA. Bead milling can also be used to split DNA from lysed cells.

Materials and methods

A representative schematic of the presently described exemplary method is provided in fig. 1. In more detail, the collection 10⁹Microbial cells were intact and pelleted by centrifugation (15000g for 5 min). The cells were then washed once with PBS and pelleted again by the same centrifugation procedure. Resuspend cells in premix buffer: 1.2mL PBS +34uL 37% formaldehyde (1% final formaldehyde concentration), and at room temperature in 30 minutes to crosslink DNA and protein. 170ul of 1M glycine solution (125mM final concentration) was added and the mixture was incubated for an additional 30 minutes to allow glycine to quench the crosslinking reaction. Then, the cells were pelleted by centrifugation (15000g for 5 min) and the supernatant was discarded. The pelleted cells can then be stored at-20 ℃ until use.

The cell pellet (. about.10. mu.l) was then resuspended in 200. mu.l of bead-milling solution (200. mu.l of 1 XTS, 2. mu.l of 100X Halt Protease Inhibitor (Thermofisiher), 2. mu.l of Triton X-100). Then, 100. mu.l of 0.5mm diameter glass beads (Qiagen) were addedIn suspension, the suspension is vortexed again at maximum speed (VWR)Vortex finder Mini 120v)3x5 minutes, with 2 minutes incubation on ice at each vortex interval. The bead milling step generates free DNA ends for the next ortho-ligation step. In contrast, most HiC preparations use restriction enzymes for the same purpose, which can be affected by genome coverage bias, lower resolution, and more complex laboratory procedures. The suspension was then centrifuged briefly to separate the glass beads. The cell lysate at this point was found to be in the supernatant. The lysate was then transferred to a new pot and centrifuged further (15000g for 5 min), after which the supernatant was discarded. The pellet was then resuspended in 500. mu.l of 1 × TBS and further centrifuged (17000g for 5 min). The supernatant was discarded and the pellet was resuspended in 200. mu. l H₂And (4) in O. The resuspended split DNA was then diluted 10-fold and its concentration was measured using a qubit (thermolfisher). Thereafter, 1 to 5. mu.g of resuspended split DNA was subjected to DNA end repair ("blunt-ended"), DNA fragments resulting from the previous bead-milling step. The reaction mixtures used for the blunt-end termination are described in table 1. The reaction was incubated at 20 ℃ for 30 minutes.

TABLE 1

1 to 5. mu.g of split DNA	xμL
		NEBNext end repair buffer 10 ×	10μL
Enzyme mixture	5μL
		H2O	85-x uL
Total of	100μL

After incubation for 30 minutes, the reaction mixture was centrifuged at maximum speed for 5 minutes on a bench top centrifuge, followed by discarding the supernatant. The pellet was resuspended in 200. mu.l of water. The resuspended end-repair DNA was then diluted 10-fold and its concentration was measured using a Qubit.

Thereafter, the end-repaired DNAs are subjected to proximity ligation. The T4 ligase ligation reaction was set to a DNA concentration of 1-2 ng/. mu.l. The reaction mixture for the T4 ligase ligation reaction is described in table 2. The reaction was incubated at room temperature for 4 hours with occasional mixing.

TABLE 2

10x T4 ligase buffer	100μL
		DNA	1-2μg
Water (W)	To 1000. mu.L
		T4 DNA ligase (NEB, 2M Unit/mL)	10μL

After an incubation time of 4 hours, the reaction mixture was centrifuged at 17000g for 5 minutes. Then 750. mu.l of the supernatant was removed without disturbing the pellet. Then 40. mu.l of 5M NaCl was added and the mixture was vortexed to resuspend the pellet. The mixture was incubated overnight at 65 ℃ to inactivate the T4 ligase and to de-crosslink. Concatemer DNA molecules should be formed after the proximity ligation step of the method. Therefore, for QC purposes, the concatemer product should be visible by agarose gel electrophoresis.

If desired, a plurality of 250. mu.l of the ligated DNA solution can now be combined. Then 5. mu.l of Triton-x100 and 50. mu.l of 10% Tween-20 were added to each 250. mu.l of the ligated DNA solution. Then water was added to make a total volume of 1 ml. To purify the DNA, 45. mu.l proteinase K solution (Qiagen) and 2. mu.l of 100mg/ml RNaseA solution were added to the ligated DNA solution and incubated at 37 ℃ for 30 minutes. Then, 350. mu.l of Qiagen buffer B2 were added to the reaction while incubating for another 30 minutes at 50 ℃.

Then, phenol: chloroform extraction and ethanol precipitation to purify the DNA, i.e., the purified DNA. The quality of the DNA was then assessed by nanodrop and agarose gel electrophoresis and quantified using Qubit.

To amplify the purified DNA, PCR template preparation was performed. The purified DNA was treated with FFPE (NEB) and Ultra-II end-prep module (NEB). The reaction mixture is described in table 3.

TABLE 3

H2DNA in O	xμL(100ng–500ng)
		NEBNext FFPE DNA repair buffer	3.5μL
NEBNext FFPE DNA repair mixture	2μL
		UltraII End-prep reaction buffer	3.5μL
UltraII End-prep enzyme mixture	3μL
		Total of	60μL

The reaction mixture was mixed, centrifuged and incubated at 20 ℃ for 15 minutes and then at 65 ℃ for 5 minutes in a thermal cycler. The reaction was purged using 0.4x SPRI beads (e.g., 24. mu.l SPRI per 60. mu.l reaction) to select DNA over 1kb in length. Then, a PCR sequencing linker compatible with the Oxford Nanopore Technologies sequencing platform was ligated to the DNA. The DNA was then purified using 0.4 XSPRI beads.

In order to obtain the optimal amount of DNA for a nanopore, the DNA sample should not have a large amount of high molecular weight amplicons. Therefore, it is suggested that the lead PCR experiment determines the optimal number of PCR cycles, first performing 8x to 12x cycles, and then visualizing these cycles by agarose gel electrophoresis. Multiple 25 μ l PCR reactions were performed as shown in Table 4.

TABLE 4

	Volume of
		DNA template	5-10ng
ONT PCR primer mixture (PRM)	0.5μL
		H2O	To 12.5. mu.L
NEB Long amplification Hot Start 2 × Mastermix	12.5μL
		Total of	25μL

For nanopore-based sequencing applications, for PCR products greater than 2 to 3kb, the size of the optimal PCR cycle DNA product is then selected by gel extraction or bluepippin (sage science). Nanopore-based sequencing is then performed according to nanopore library preparation and sequencing protocols known in the art.

Bioinformatics workflow

Fig. 2 provides a representative schematic of a bioinformatic analysis workflow suitable for use in the presently described exemplary method. In any of the described methods, the bioinformatic analysis workflow can be used to obtain metagenomic contact maps from sequencing data derived using methods applied to metagenomic samples. In more detail, nanopore sequencing data is generated by the methods described earlier to provide nanopore sequencing reads. Reads are first aligned to a collection of reference sequences for chromosomal and extra-chromosomal sequences (e.g., plasmids) using BWA-SW (Li H. and Durbin R. (2010) Fast and acid long-reading alignment with Burrows-Wheeler. Transform. Bioinformatics, Epub. [ PMID: 20080505 ]). The reads of each alignment are filtered to retain the smallest set of alignments that traverse most of the reads. The reference genome is then divided into equally sized bins, one for each aligned fragment of the nanopore sequencing read. Finally, the total number of bin-to-bin contacts was calculated from all nanopore sequencing reads and visualized in the contact map. Extrachromosomal elements can be assigned to their hosts by determining which chromosomes have the most exposure to the element.

Results

The results shown in fig. 3 demonstrate that the results produced by the above-described methods and workflows demonstrate the identification of intrachromosomal and extrachromosomal contacts in probiotic samples. Genomic DNA from probiotic food supplement samples, which contained 15 known bacterial strains (fig. 3A), were applied to the above-described methods and workflow, and nanopore sequencing data was generated. Contact maps of bacterial chromosomes and plasmids within the samples were prepared according to the bioinformatics workflow above (fig. 3B and 3C). The mean nucleotide identity plot (fig. 3D) revealed low levels of spurious interactions between species, most likely due to ambiguity in the mapping of nanopore sequencing reads. Fig. 3E and 3F summarize the contacts of each bacterial chromosome. The plasmid associates with the expected host genome and recognizes the intrachromosomal interactions, which are valuable for binning and assembly of contact maps.

Example 2

This example describes another exemplary laboratory workflow applicable to the present disclosure, namely a method for determining interactions between intracellular elements. In particular, the method is used to study interactions between genomic elements that are not adjacent in the primary sequence. In addition to simplifying assembly, the disclosed methods provide a way to associate a plasmid with its host genome when applied to a metagenomic sample. An alternative approach for determining interactions between one or more elements within a DNA molecule employs restriction enzyme cleavage of the cross-linked DNA prior to performing proximity ligation. However, restriction enzyme cleavage is rather time consuming and the choice of restriction enzyme is influenced by the nucleotide composition of the genome in the sample, which is not always known in advance, especially when performing metagenomic studies. The present disclosure provides a method to avoid restriction enzyme cleavage by using mechanical disruption (e.g., bead milling) to simultaneously lyse cells and divide DNA. Bead milling can also be used to split DNA from lysed cells.

Materials and methods

Sample Collection and Cross-linking

Collection and isolation of large 2-3X 10 by methods known in the art⁹And (4) intact bacterial cells. Cells were resuspended in pre-mixed cross-linking buffer (1.2mL PBS, 34 μ L37% formaldehyde (1% formaldehyde final concentration)) and incubated for 30 minutes at room temperature with occasional mixing. L of 1M glycine (125mM glycine final concentration) was added at 170 μ M to quench the crosslinking reaction, and the sample was incubated at room temperature for an additional 20 minutes. The sample was then centrifuged at 17000g for 5 minutes, the supernatant discarded, and the crosslinked cell pellet (fixed cells) was washed with 1 × TBS. The sample was centrifuged at 17000g for an additional 5 minutes and the supernatant discarded. The precipitate can then be stored at-80 ℃ for further use.

Cell lysis and end repair

If it was previously stored at-80 ℃, the pellet of fixed cells should be thawed on ice. The cells (approximately 10. mu.L) were then resuspended in 200. mu.L of bead mill solution, as shown in Table 5. The total volume of the bead milling solution can be scaled up/down as desired.

TABLE 5

Reagent	Volume of
		1X TBS	200μL
100X arrestin inhibitors	4μL
		Triton X-100	2μL
Total of	206μL

Then 100 μ L of 0.5mm diameter glass beads (Qiagen) were added to the resuspended cells, and the sample was vortexed at the highest speed for 3X5 minutes, every five minutes for 2 minutes on ice. The sample was briefly centrifuged to eliminate air bubbles and the sample was remixed by blowing the sample solution up and down to produce a homogenous cell lysate. The lysate was then transferred to a new tube and centrifuged at 17000g for 5 minutes. The supernatant was discarded and the pellet was resuspended in 500. mu.L of 1 XTSS. The sample was centrifuged again at 17000g for 5 minutes and the supernatant was discarded. Resuspend the pellet in 50. mu. L H₂O, mixed well, and the DNA concentration is preferably measured by qubit (thermofisher scientific). The end-repair reactions were formulated as shown in table 6.

TABLE 6

Reagent	Volume of
		Up to 5. mu.g of chromatin DNA	xμL
NEBNext end repair buffer (10X)	10μL
		NEBNext end repair enzyme mixture	5μL
H2O	85-xμL
		Total of	100μL

The end repair reaction was incubated at 20 ℃ for 30 minutes and then centrifuged at 17000g for five minutes. The supernatant was discarded and the pellet was washed with 1 × TBS. The sample was then briefly centrifuged at 17000g, followed by discarding the supernatant. The precipitate may then be stored at-20 ℃ for future use.

Ortho-position ligation

Resuspend the pellet of end-repaired DNA in 200. mu. L H₂O and quantified by Qubit. The T4 connection was then established as shown in table 7 below.

TABLE 7

Reagent	Volume of
		H2O	1618-xμL
H20.9ug DNA in O	xμL
		10X T4 ligase buffer (Thermofeisher)	180μL
Mixing thoroughly
		T4 DNA ligase (Thermofisiher, 30weissU/uL)	6μL
Total of	1800μL

The final concentration of DNA in the proximity ligation reaction was 0.5 ng/. mu.L. The reaction was incubated at 22 ℃ for four hours with occasional mixing. The sample was then centrifuged at 17000g for 5 minutes. 1375. mu.L of the supernatant was removed, leaving approximately 475. mu.L of the supernatant. 25 μ L of 5M NaCl was added to a final volume of 500 μ L. If desired, multiple 500 μ L reactions can now be combined in the same sample tube. The samples were then incubated overnight to effect decrosslinking.

Purification

For every 500. mu.L of decrosslinking reaction, the reagents listed in Table 8 were added in order.

TABLE 8

The reaction was incubated at 37 ℃ for 30 minutes. 170 μ L of Qiagen buffer B2 was added to each reaction described above, and the reactions were incubated at 50 ℃ for 30 minutes. The DNA was then purified by phenol chloroform isoamyl alcohol and subsequent isopropanol precipitation.

Sequencing

Purified DNA can be 1) prepared directly for sequencing using standard library preparation workflows/kits of Oxford Nanopore Technologies (which enables natural DNA modifications (epigenomics) to be retained in the sequencing data generated); or 2) by PCR amplification using the PCR sequencing workflow/kit of Oxford Nanopore Technologies.

For nanopore-based sequencing applications, for PCR products greater than 2 to 3kb, the size of the optimal PCR cycle DNA product is then selected by gel extraction or bluepippin (sage science). Nanopore-based sequencing is then performed according to nanopore library preparation and sequencing protocols known in the art.

Bioinformatics workflow

Fig. 2 provides a representative schematic of a bioinformatic analysis workflow suitable for use in the presently described exemplary method. In any of the described methods, the bioinformatic analysis workflow can be used to obtain metagenomic contact maps from sequencing data derived using methods applied to metagenomic samples. In more detail, nanopore sequencing data is generated by the methods described earlier to provide nanopore sequencing reads. Reads are first aligned to a collection of reference sequences for chromosomal and extra-chromosomal sequences (e.g., plasmids) using BWA-SW (Li H. and Durbin R. (2010) Fast and acid long-reading alignment with Burrows-Wheeler. Transform. Bioinformatics, Epub. [ PMID: 20080505 ]). The reads of each alignment are filtered to retain the smallest set of alignments that traverse most of the reads. The reference genome is then divided into equal-sized bins, one for each aligned fragment of the nanopore sequencing read. Finally, the total number of bin-to-bin contacts was calculated from all nanopore sequencing reads and visualized in the contact map. Extrachromosomal elements can be assigned to their hosts by determining which chromosomes have the most exposure to the element.