Novel method

Abstract

The present invention provides a method of preparing eukaryotic cellular material from a mucosal sample, which comprises a mixture of mammalian and prokaryotic cells. The method comprises the steps of: (i) obtaining the mucosal sample; (ii) preferentially lysing the mammalian cells in the mixture into a soluble state; (iii) separating the lysed soluble cellular material from non-lysed prokaryotic material; and (iv) removing any prokaryotic material in the lysed solution cellular material. The method yields a sample of mammalian cellular material that is substantially free of prokaryotic cells and / or cellular material. Also provided are methods of diagnosing a disease or disorder and treating such diseases / disorders, comprising performing the preparation method described herein and analysing the sample of mammalian cellular material for a marker associated with the disease or disorder.

Description

[0001] ORI-C-P3671 PCT

[0002] NOVEL METHOD

[0003] FIELD OF THE INVENTION

[0004] The present invention provides a method of preparing eukaryotic cellular material from a mucosal sample, which comprises a mixture of mammalian and prokaryotic cells. The method comprises the steps of: (i) obtaining the mucosal sample; (ii) preferentially lysing the mammalian cells in the mixture into a soluble state; (iii) separating the lysed soluble cellular material from non-lysed prokaryotic material; and (iv) removing any prokaryotic material in the lysed solution cellular material. The method yields a sample of mammalian cellular material that is substantially free of prokaryotic cells and / or cellular material. Also provided are methods of diagnosing a disease or disorder and treating such diseases / disorders, comprising performing the preparation method described herein and analysing the sample of mammalian cellular material for a marker associated with the disease or disorder.

[0005] BACKGROUND OF THE INVENTION

[0006] Mucosal membranes line the epithelial cell layer of bodily cavities in select organisms. The mucosa is connected to both the circulatory and lymphatic systems and is structured in three layers; an inner epithelium, a middle layer of lamina propria, and an outer layer (muscularis mucosae) that separates the mucosa from submucosa.

[0007] The mucosal membrane has vast and diverse roles, from acting as a barrier for physical and biological protection, to maintaining homeostasis, as well as sensory perception. The complexity of mucosal function is reflected in its highly varied contents including, without limitation, ‘peptide shields’, local secretions (such as immunoglobulins), antimicrobial substances, iron-binding proteins and the microbiota. The mucus ‘stream’ can be perceived as a local circulatory system that facilitates general biological function and activities, where changes in the content, proportions and nature of the mucus are highly reflective of the associated microenvironment. As the barrier between the body and the environment, samples taken from mucosal membranes comprise a mixture of cells and cellular material from the organism and cells / cellular material not derived from the organism themselves. Such nonorganism derived cells / cellular material is often prokaryotic (e.g. bacterial).

[0008] There is therefore a need to develop methods of separating mammalian (e.g. human) cellular material, in particular mammalian nucleic acids, from samples containing a mixture of mammalian and prokaryotic (e.g. bacterial) cells and cellular material. Such methods would find particular utility in separating mammalian cellular material from mucosal samples, ORI-C-P3671 PCT including human colorectal and vaginal mucosal samples, which comprise a complex mixture of human and bacterial cells.

[0009] SUMMARY OF THE INVENTION

[0010] According to a first aspect of the invention, there is provided a method of preparing mammalian cellular material from a mucosal sample, comprising the steps of:

[0011] (i) obtaining a mucosal sample comprising a mixture of mammalian and pro / karyotic cells;

[0012] (ii) preferentially lysing the mammalian cells in the mixture into a soluble state while retaining substantially all of the prokaryotic cells in a non-lysed insoluble state;

[0013] (iii) separating the lysed soluble cellular material from the non-lysed insoluble prokaryotic cells; and

[0014] (iv) removing any prokaryotic cellular material in the lysed soluble cellular material using one or more prokaryotic-specific marker, thereby yielding a sample of mammalian cellular material with reduced prokaryotic cells and / or prokaryotic cellular material compared to the obtained mucosal sample.

[0015] In some embodiments, the method additionally comprises the step of:

[0016] (ib) separating any soluble cellular material from the mucosal sample prior to preferentially lysing the mammalian cells in step (ii), and adding the separated soluble cellular material to the lysed soluble cellular material prior to step (iv).

[0017] In particular embodiments, removing step (iv) is by negative selection, such as wherein the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic genetic material and, for example, the one or more prokaryotic-specific marker is bacterial-specific methylation of DNA, such as DNA adenine methylation. In further embodiments, the methylated bacterial DNA is digested in step (iv) using a methylationspecific or methylation-dependent restriction enzyme.

[0018] In further embodiments, the mucosal sample is a colorectal mucosal sample, in particular a human colorectal sample comprising a mixture of human and bacterial cells. In yet further embodiments, the mucosal sample is not diluted prior to step (ii), and / or cells in the mucosal sample are not pre-purified prior to step (ii), in particular wherein mammalian cells in the mucosal sample are not pre-purified prior to step (ii).

[0019] In still further embodiments, the method further comprises the step of: ORI-C-P3671 PCT

[0020] (v) analysing the sample of mammalian cellular material, optionally by sequencing wherein the mammalian cellular material is genetic material, such as DNA and / or RNA.

[0021] In further embodiments, the sample is analysed to detect the presence or absence of a marker associated with a disease or disorder.

[0022] In a further aspect of the invention, there is provided a method of diagnosing a disease or disorder in a mammalian subject, the method comprising obtaining a sample of mammalian cellular material according to the method described herein and analysing said sample for a marker associated with the disease or disorder, such that presence of the marker indicates a likelihood that the subject is suffering from the disease or disorder and absence of the marker indicates that the subject is unlikely to be suffering from the disease or disorder.

[0023] In a yet further aspect, there is provided a method of treating a disease or disorder in a mammalian subject in need thereof, said method comprising the steps of:

[0024] (i) performing the method of diagnosing as described herein on a mucosal sample obtained from the subject; and

[0025] (ii) administering to the subject a treatment for the disease or disorder if the disease / disorder-associated marker is present.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] Figure 1 : Antibody mediated pulldown of bacterial DNA / associated binding proteins. Percentage of human and bacterial mass calculated for each sample (n = 3; A: sample 1 , B: sample 2, C: sample 3), with bulk extraction methods (“Original”), compared to the positive and negative antibody pulldown fractions obtained from optimised purification method 1 as described in Example 2. This method increases the purity of the human fraction by approx. 10-15%, with 70-90% of total bacterial mass being lost in the positive fraction.

[0028] Figure 2: Specific endonuclease digestion of bacterial DNA and size specific purification. Percentage of human and bacterial mass calculated for each sample (n = 2; A: sample 1 , B: sample 2), with bulk extraction methods (“Control”), compared to the percentage recovery of bacterial and human DNA in the SPRI size selection positive and negative fractions obtained from optimised purification method 2 as described in Example 3. This method increases the purity of the human fraction by up to approx. 30%.

[0029] Figure 3: Negative selection of human DNA by hybridisation capture of generic bacterial regions. Percentage of human and bacterial mass calculated for each sample (n = 3), with bulk extraction methods (“Exsig Mag”), compared to the percentage recovery of bacterial and human DNA from the negative selection hybridisation enrichment method ORI-C-P3671 PCT

[0030] (method 3 as described in Example 4). This experimentation is a proof of concept, detailing the negative selection of human DNA, by the targeted removal of representative conserved sections of the bacterial genome.

[0031] Figure 4: Non-CpG conversion rate of human DNA in extracted samples. The non-CpG conversion rate (labelled “CpG”) was determined from multiple independent samples extracted in a conventional manner and compared to those enriched by method 1 (Example 2) and method 2 (Example 3) herein. In order for samples to be reliably analysable, conversion rate must be above 98%. The 10 samples assessed all failed to meet this criteria without enhanced enrichment, but 8 out of the 10 samples achieved conversion rates above 98% when enriched with either method 1 or method 2.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention and disclosures herein address a need for separating mammalian (e.g. human) cellular material, in particular nucleic acids, from samples containing a mixture of mammalian and prokaryotic cells and cellular material, such as human colorectal mucosal samples comprising a complex mixture of human and bacterial cells, and are based on the discovery that mammalian cells can be preferentially lysed compared to prokaryotic / bacterial cells within said sample. Furthermore, the invention recognises and addresses a need to further remove any prokaryotic cellular material that may be present in the lysed soluble fraction after preferential lysis. The inventors are not aware of any such methods comprising preferential lysis of mammalian cells followed by removal of prokaryotic cellular material in the lysed soluble fraction having been disclosed in the art to date.

[0034] For example, Charalampous et al. (2019) Nature Biotecnol., 37(7):793-792 (doi: https: / / do: org / 10.1038 / S4 587-019-0156-5) describes preferentially lysing host cells from human respiratory samples (sputum, endotracheal secretions, bronchoalveolar lavage and endotracheal aspirate) to prepare a sample of bacterial cells in which host-DNA and host-cells have been depleted and from which bacterial DNA is subsequently extracted. In the method of Charalampous etal. host (human) cells are preferentially lysed using saponin and free DNA is digested before lysing the bacteria. Thus, there is no free bacterial cellular material following the preferential lysis step and only after final bacterial lysis is this released into suspension. As such, only whole bacteria which are not lysed prior to preferential lysis are recovered in Charalampous et al. and any which may already be lysed (e.g. prior to or during sample collection or during sample transport or handling) are lost, leading to possible loss of sample diversity. Furthermore, the method of Charalampous et al. yields purified bacterial DNA from a human mucosal sample and is thus unsuitable for the present objective to prepare a sample of mammalian cellular material substantially free of prokaryotic cells and / or cellular material. ORI-C-P3671 PCT

[0035] This is contrary to the findings of the present inventors which is that some free (i.e. soluble) bacterial cellular material will be present before / after mammalian cell preferential lysis and thus needs to be removed before mammalian cellular material can be purified from the soluble fraction. Such removal is provided in step (iv) of the method described herein, which combines said removal with preferential lysis of mammalian cells in step (ii) herein to yield a sample of mammalian cellular material substantially free of prokaryotic cells and / or cellular material. Such removal also reduces a possible loss of sample diversity in the prokaryotic cells and / or cellular material in embodiments where the method is used as described herein to yield a sample of prokaryotic cells and / or cellular material substantially free of mammalian cells and / or mammalian cellular material.

[0036] Thus, according to a first aspect of the invention there is provided a method of preparing mammalian cellular material from a mucosal sample, comprising the steps of:

[0037] (i) obtaining a mucosal sample comprising a mixture of mammalian and prokaryotic cells;

[0038] (ii) preferentially lysing the mammalian cells in the mixture into a soluble state while retaining substantially all of the prokaryotic cells in a non-lysed insoluble state;

[0039] (iii) separating the lysed soluble cellular material from the non-lysed insoluble prokaryotic cells; and

[0040] (iv) removing any prokaryotic cellular material in the lysed soluble cellular material using one or more prokaryotic-specific marker, thereby yielding a sample of mammalian cellular material with reduced prokaryotic cells and / or prokaryotic cellular material compared to the obtained mucosal sample.

[0041] As will be appreciated from the context herein, the term “preparing” may be used interchangeably with “separating” (such as “separating from”), “purify”, “enriching” and the like. Such preparing / separating may also be considered a “cleaning” or “decontamination” of the mammalian cellular material from prokaryotic cells and / or cellular material which is present in the sample as part of a mixture. Thus, according to the method described herein the mammalian cellular material is prepared or separated from the mixture of mammalian and prokaryotic cells comprised in a sample, such that said prepared / separated mammalian cellular material can be considered purified / enriched from said mixture, purified / enriched from prokaryotic cells and / or cellular material in the mixture and / or cleaned / decontaminated of prokaryotic cells / cellular material in the mixture. ORI-C-P3671 PCT

[0042] However, as will further be readily appreciated, the method described herein may also yield “prepared”, “separated” or “enriched” prokaryotic cells and / or cellular material, by virtue of preparing / separating the mammalian cellular material present in the mixture comprised in the sample. Thus, in some embodiments the method described herein yields a sample of prokaryotic cells and / or cellular material with reduced mammalian cells and / or mammalian cellular material compared to the starting material. According to these embodiments, the prokaryotic cells and / or cellular material are collected as the non-lysed insoluble fraction in step (iii) and as the removed prokaryotic cellular material in step (iv). Thus, in one embodiment wherein the method is used to prepare / separate prokaryotic cells and / or cellular material, removal in step (iv) is by positive selection of the one or more prokaryotic-specific marker.

[0043] Thus, in another aspect of the invention there is provided a method of preparing prokaryotic cells and / or cellular material from a mucosal sample, comprising the steps of:

[0044] (i) obtaining a mucosal sample comprising a mixture of mammalian and prokaryotic cells;

[0045] (ii) preferentially lysing the mammalian cells in the mixture into a soluble state while retaining substantially all of the prokaryotic cells in a non-lysed insoluble state;

[0046] (iii) separating the lysed soluble cellular material from the non-lysed insoluble prokaryotic cells; and

[0047] (iv) removing any prokaryotic cellular material from the lysed soluble cellular material by positive selection using one or more prokaryotic-specific marker, optionally lysing the insoluble prokaryotic cells and combining the lysate with the removed prokaryotic cellular material of step (iv), thereby yielding a sample of prokaryotic cells and / or cellular material with reduced mammalian cellular material compared to the obtained mucosal sample.

[0048] The method described herein comprises step (i) of obtaining a mucosal sample comprising a mixture of mammalian and prokaryotic cells. In some embodiments, “obtaining” as used herein refers to the collection of the mucosal sample from a mammalian subject, wherein such obtaining may include active collection from the subject, for example by non-invasive methods such as skin surface collection, or by non- / minimally invasive methods such as oral, nasal, aural, ocular surface, vaginal, penile, urethral or rectal / colorectal swab / collection. Invasive methods of obtaining a sample would not commonly be used in the methods herein as they would unlikely provide a sample comprising a mixture of mammalian and prokaryotic cells. However, in situations where a sample obtained invasively may comprise a mixture of mammalian and prokaryotic cells (e.g. in cases of sepsis / potential sepsis), “obtaining” may include invasive methods of sample collection such as venepuncture and other methods of ORI-C-P3671 PCT blood collection. Certain methods of vaginal, penile, urethral, bronchial, alveolar or rectal / colorectal sample collection may also be considered invasive and are included herein by virtue of providing a mucosal sample. In other embodiments, “obtaining” refers to providing a sample which has previously been collected from the mammalian subject, said collection not encompassed by the invention, i.e. wherein the method described herein does not comprise any methods performed on the human or animal body or in vivo. Thus, according to these embodiments “obtaining” may be used interchangeably with “providing”, such as “providing a mucosal sample obtained from a mammalian subject”. Alternatively, step (i) may not comprise active collection from the subject. Thus, in other embodiments step (i) comprises providing a mucosal sample comprising a mixture of mammalian and prokaryotic cells which has been obtained from a subject. Such prior obtaining may be by any method previously described herein.

[0049] A mucosal sample “obtained” or “provided” herein comprises a sample collected from a mucosal surface or membrane of a mammalian subject. In some embodiments, the mammalian subject is a human. Thus, in further embodiments the mucosal sample is obtained from a human subject, such as from a human mucosal surface / membrane or mucosa. Mucosal surfaces / membranes or mucosa line the cavities of the mammalian body and provide barriers between the interior of the body (e.g. the internal organs) and the outside environment, and mucosal samples taken from them thus often comprise a mixture of cells from the mammalian subject and cells / cellular material from the surrounding environment, such as prokaryotic cells (e.g. bacteria). They comprise one or more layers of epithelial cells and connective tissue, and develop from the endoderm during embryonic development. A particular example for the purpose of the present invention is the gastrointestinal tract, including the rectal cavity and / or intestines. Thus, in one embodiment the mucosal sample is a colorectal mucosal sample. In a further embodiment, the mucosal sample is from the anal canal and / or rectal cavity. Mucosal samples taken from the rectal cavity, anal canal and / or intestines, in particular the colon or large intestine, will comprise a complex mixture of cells from the mammalian subject and bacteria (i.e. prokaryotic cells). For example, a typical human stool / faeces excreted from the rectum contains a mixture of bacterial biomass, protein or nitrogenous matter, carbohydrate or undigested plant matter, fat, insoluble calcium and iron phosphate salts, intestinal secretions, mucus and shed epithelial cells. Thus, a mucosal sample obtained from the lower gastrointestinal tract, such as from the colon and in particular from the rectal / colorectal cavity and / or anal canal, will comprise a mixture of cells from the organism from which the sample is obtained (i.e. a mammal herein), mucus and bacteria, and may further comprise any of the other above constituents of stool / faeces. Thus, in a yet further embodiment the mucosal sample is a mammalian colorectal sample comprising a mixture of ORI-C-P3671 PCT mammalian and bacterial (i.e. prokaryotic) cells. In a particular embodiment, the mucosal sample is a human colorectal sample comprising a mixture of human and bacterial cells. Further examples of mucosal surfaces / mem branes and mucosa include: the respiratory tract and respiratory mucosa, including the nasal cavity and mucosa, pharynx, larynx, trachea, bronchi, primary and secondary bronchus, tertiary bronchi, bronchioles and alveoli; endometrium, including the mucosa of the uterus; vaginal mucosa; penile mucosa; urethral mucosa; nasal and olfactory mucosa; the oral cavity, including the throat, alveolar, buccal, labial and masticatory mucosa, and the frenulum of the tongue; and conjunctiva of the eye.

[0050] In certain embodiments, the mucosal sample is obtained using a cell sampling device. An example of a suitable cell sampling device is that disclosed in WO 2006 / 003447, which is hereby specifically incorporated by reference. Such a device is suitable for colorectal sampling and of other mucosal surfaces of the body, such as sampling the vaginal mucosa. Thus, in one embodiment the mucosal sample is obtained using a colorectal cell sampling device. In a further embodiment, the colorectal sampling device is that disclosed in WO 2006 / 003447. Wherein the mucosal sample is a colorectal mucosal sample collected using a cell sampling device such as that of WO 2006 / 003447, the method described herein is suitable for preparing mammalian cellular material from said sample without any dilution or pre-filtering prior to step (ii). Additionally, the method described herein requires no prepurification prior to step (ii), in particular no pre-purification of mammalian cells in the mucosal sample is required prior to step (ii). This is in contrast to methods previously described in the art, in which the composition of colorectal mucosal samples requires dilution, filtering or other sample preparation prior to any preparation / separation method steps (e.g. in Charalampous et al. respiratory samples are centrifuged and the pelleted cells are resuspended in PBS prior to preferential lysis). Without being bound by any particular theory, it is believed that the dilution, filtering and other sample preparations performed in the methods previously described in the art may be used to yield a starting sample with known consistency. This may be important in previously described methods due to the very high variability of colorectal mucosal samples, which can have high viscosity due to a large amount of ‘thick’ mucus or a low viscosity and thus be considered ‘thin’ or ‘watery’. The present method can be performed on such colorectal mucosal samples with highly variable consistency / viscosity. Thus, in one embodiment the mucosal sample is not diluted prior to step (ii). In a further embodiment, the mucosal sample is not filtered prior to step (ii). In a yet further embodiment, cells in the mucosal sample are not pre-purified prior to step (ii). In a still further embodiment, mammalian cells in the mucosal sample are not pre-purified prior to step (ii). The advantages of the method described herein requiring fewer or no sample preparation steps prior to step (ii) include, without limitation: less handling of a biological sample, resulting in improved biosafety; ORI-C-P3671 PCT increased cellular yield due to less sample handling and e.g. fewer washes; reduced loss of cellular material due to e.g. filtering and pre-purification; quicker completion time due to fewer steps; decreased costs due to fewer steps, fewer reagents being required and quicker completion time; and decreased potential loss of sample diversity due to loss of cellular material e.g. due to washing.

[0051] As described, a mucosal sample obtained from a mammal herein will comprise a mixture of cells originating from said mammal (e.g. epithelial cells) and prokaryotic cells (i.e. bacteria). The object of the present invention is to prepare / separate as described hereinbefore the mammalian cellular material from cells and cellular material of bacterial origin. In one embodiment, the mammalian cellular material is from epithelial cells. Thus, in a further embodiment the mammalian cellular material is epithelial cellular material. In yet further embodiments, the mammalian cellular material is from other colonic cell types, such as enterocytes (absorptive cells), goblet cells, enteroendocrine cells, Paneth cells, epithelial / mucosal stem cells, M cells, mesenchymal cells, and immune / inflammatory cells, including mast cells, eosinophils, basophils, macrophages, neutrophils, muscularis mucosae and propria cells, stromal cells, adipose cells, ganglion cells, enteric glial cells, and lymphocytes (e.g. B cells, lamina propria plasma cells, intraepithelial and lamina propria T cells). Thus, in some embodiments the mammalian cellular material is cellular material from a colonic cell type described herein. A resulting sample of mammalian cellular material with reduced prokaryotic cells and / or cellular material compared to in the starting sample can be utilised to investigate certain conditions of the mammalian subject from which the cellular material originates, without / with reduced contaminating prokaryotic cells or cellular material. This is particularly useful when analysing genetic material within the cellular material, the origin of which may not be easily determined from sequence alone (i.e. its origin is, in part, determined from the type of sample, the location from which it was obtained and / or its processing, such as described herein). Thus, a sample of mammalian cellular material with reduced prokaryotic cells and / or cellular material is useful when analysing any cellular material of which the origin may not be easily determined solely from the identity or properties of the material itself, e.g. when its full identity is determined, at least in part, from the sample from which the cellular material was obtained, the location where the sample was obtained and / or the processing (e.g. purification / separation) of the cellular material prior to analysis, such as by the method described herein. In certain embodiments, the method further comprises step (v) of analysing the sample of mammalian cellular material. Such investigations / analysis can comprise analysis of genetic material and nucleic acids within the mammalian cellular material, for example by PCR including quantitative, real-time and reverse transcription PCR to identify the presence of particular sequences (e.g. the presence of particular genomic ORI-C-P3671 PCT sequences / mutations or the presence / abundance of particular transcripts) and by sequencing. Thus, in one embodiment the sample of mammalian cellular material is analysed by sequencing, in particular wherein the mammalian cellular material is genetic material. In a further embodiment, the sample of mammalian cellular material is analysed by PCR, such as qPCR or rt-PCR. Such investigations / analysis can also comprise analysis of proteins within the mammalian cellular material. Thus, in a yet further embodiment the sample of mammalian cellular material is analysed by mass-spectrometry, Western Blot and the like.

[0052] Thus, in some embodiments the mammalian cellular material comprises genetic material and / or nucleic acids. In further embodiments, the genetic material is DNA and / or RNA. In yet further embodiments, the nucleic acids comprise genomic sequences, transcriptom ic sequences (e.g. mRNA) and / or non-coding sequences. In a particular embodiment, the mammalian cellular material comprises genomic nucleic acid sequences from a mammalian subject. In some embodiments, the mammal is a human. Thus, in a further embodiment the mammalian cellular material is human genetic material and / or nucleic acid, in particular human genomic nucleic acid sequences. According to these embodiments, the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic genetic material as described further herein.

[0053] However, as will be readily appreciated, the method described herein will find utility in preparing / separating any mammalian cellular material. Thus, in further embodiments the mammalian cellular material comprises proteins. Such proteins are of mammalian origin. In a yet further embodiment, the mammalian cellular material is human cellular protein. In still further embodiments, the mammalian cellular material comprises both genetic material and protein, such as human cellular protein and human genetic material and / or nucleic acid, in particular human genomic nucleic acid sequences. According to these embodiments, the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic protein as described further herein.

[0054] The term “substantially free” as may be used herein refers to an absence of prokaryotic cells and / or cellular material at a level which may be reasonably expected by a skilled person in the art. For example, the method may yield a sample of mammalian cellular material that is at least 90% free, 91 % free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98% free, 99% free or 100% free of prokaryotic cells and / or cellular material, or vice versa. Alternatively, the method may yield a sample of mammalian cellular material comprising none, 1 % or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less or 10% or less of prokaryotic cells and / or cellular material, or vice versa. The term ORI-C-P3671 PCT

[0055] “reduced” is used herein in its normal meaning and as would be readily understood by the skilled person. Specifically, the amount of prokaryotic cells and / or cellular material may be reduced in the sample of mammalian cellular material following step (iv) herein compared to the amount of prokaryotic cells and / or cellular material in the starting material, i.e. in the mucosal sample comprising a mixture of mammalian and prokaryotic cells obtained / provided in step (i) of the method described herein. Such reduction may be in the number of prokaryotic cells, in the copy number of bacterial genomes and / or in the amount of prokaryotic cellular material, such as the mass of prokaryotic cellular material. For example, the method may yield a sample of mammalian cellular material that has about 10% reduced, about 20% reduced, about 30% reduced, about 40% reduced, about 50% reduced, about 60% reduced, about 70% reduced, about 80% reduced, about 90% reduced or more of prokaryotic cells and / or cellular material. As demonstrated herein, the present method can reduce the mass of prokaryotic cells and / or cellular material by approx. 77%, approx. 89%, approx. 93% (see Example 2), approx. 15% and approx. 21% (see Example 3) in various tested samples. Therefore, in certain embodiments the prokaryotic cells and / or cellular material is reduced by greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 30%, greater than or equal to about 40%, greater than or equal to about 50%, greater than or equal to about 60%, greater than or equal to about 70%, greater than or equal to about 75%, greater than or equal to about 80%, greater than or equal to about 85%, greater than or equal to about 90% or greater than or equal to about 95%. At the same time, the amount of mammalian cellular material in the sample following step (iv) herein will be increased in proportion of the total cellular material, compared to the proportion of mammalian cellular material in the obtained mucosal sample. For example, the method may yield a sample of mammalian cellular material that has about 3% increased, about 5% increased, about 10% increased, about 15% increased, about 20% increased, about 25% increased, about 30% increased or more proportion of mammalian cellular material compared to in the obtained mucosal sample. As demonstrated herein, the present method can increase the proportion of mammalian cellular material mass by approx. 3%, approx. 8%, approx. 10% (see Example 2) and approx. 30% (see Example 3) in various tested samples. Therefore, in certain embodiments the proportion of mammalian cellular material is between about 1% and 5% increased, about 3% increased, about 5% increased, between about 5% and 10% increased, between about 8% and 10% increased, about 10% increased, between about 10% and 20% increased, between about 20% and 30% increased or about 30% increased or more compared to the proportion of mammalian cellular material in the obtained mucosal sample.

[0056] The reductions described hereinbefore may also be of the mammalian cellular material in embodiments wherein the method is used to yield a sample of prokaryotic cells and / or cellular ORI-C-P3671 PCT material with reduced mammalian cells and / or mammalian cellular material as described hereinbefore. According to these embodiments, the reduction may be in the number of mammalian cells and / or the amount of mammalian cellular material, such as the mass of mammalian cellular material. For example, the method may yield a sample of prokaryotic cells and / or cellular material that has about 10% reduced, about 20% reduced, about 30% reduced, about 40% reduced, about 50% reduced, about 60% reduced, about 70% reduced, about 80% reduced, about 90% reduced or more of mammalian cellular material.

[0057] While certain values are described herein for the reduction of prokaryotic cells and / or cellular material or for the increased proportion of mammalian cellular material, and vice versa, it will be readily appreciated that the amount is dependent on any subsequent use, analysis or processing of the sample yielded following step (iv), such as the method of analysing the sample in step (v) herein. For example and as demonstrated herein, reductions in prokaryotic cells and / or cellular material and increases in the proportion of mammalian cellular material achieved by the present method provide a significantly increased likelihood of a CpG conversion rate of over 98% in epigenetic methylation analyses. Other reductions and increases suitable for other analysis methods will be readily identifiable by the skilled person.

[0058] In some embodiments, the method additionally comprises step (ib) of separating any soluble cellular material from the mucosal sample prior to preferentially lysing the mammalian cells in step (ii). The separation of soluble material prior to step (ii) ensures that any cellular material already in solution prior to any preferential lysis, such as because of lysis that has occurred prior to or during sample collection or because of lysis during sample transport or handling, is retained in the method and not lost due to washes etc. prior to preferential lysis. However, since the separated soluble cellular material may comprise both prokaryotic as well as mammalian cellular material, the soluble cellular material separated in optional step (ib) is added to the lysed soluble material prior to step (iv). Thus, according to one embodiment the method additionally comprises step (ib) of separating any soluble cellular material from the mucosal sample prior to preferentially lysing the mammalian cells in step (ii), and adding the separated soluble cellular material to the lysed soluble cellular material prior to step (iv).

[0059] The method comprises step (ii) of preferentially lysing the mammalian cells in the mixture into a soluble state. As used herein, the term “preferentially” and the like is used in its normal context and meaning, namely that the lysis of mammalian cells occurs with greater likelihood and / or to a greater amount compared to the lysis of prokaryotic cells in the mixture. Thus, mammalian cells are preferentially lysed in step (ii) while a significant amount and / or substantially all of the prokaryotic cells are retained in a non-lysed insoluble state. Preferential ORI-C-P3671 PCT lysis is achieved by virtue of mammalian cells having a structure more susceptible than prokaryotic cells to lysis by mild lysis reagents. Without being bound by theory, this is believed to be due to the presence of a cell wall in prokaryotes which provides resistance to changes in osmolarity and osmotic shock, a structure not present in mammalian cells. Thus, a lysis reagent that alters the osmolarity of the environment surrounding the cell, changes the cell’s ability to control osmolarity or leads to osmotic shock (e.g. a high salt concentration which leads to water loss from inside the cell, detergents, surfactants and cell membrane permeabilising agents, such as toxins, that compromise the cell membrane and lead to ingress of water into the cell) will preferentially lyse mammalian cells. As will be appreciated, eukaryotic cells comprising a cell wall or which are resistant to osmotic changes cannot be preferentially lysed as described herein (e.g. plant cells and / or fungi). Thus, the method described herein is specifically for separating mammalian cells in a mixture comprising prokaryotic cells and is not suitable for separating any / all types of eukaryotic cell (i.e. plant cells or fungi). The term “significant amount” in this context refers to a retention of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more or about 85% or more of the prokaryotic cells in a non-lysed insoluble state, specifically wherein between about 65% and 70% of prokaryotic cells are retained in a non-lysed insoluble state. Alternatively, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less or about 15% or less of the prokaryotic cells in the mixture are lysed into a soluble state, in particular wherein between about 30% and 35% of the prokaryotic cells are lysed into a soluble state. The term “substantially all” in this context refers to a retention of about 90% or more, about 91 % or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more or about 100% of the prokaryotic cells in a non-lysed insoluble state. Alternatively, none, about 1 % or less, about 2% or less, about 3% or less, about 4% or less, about 5% or less, about 6% or less, about 7% or less, about 8% or less, about 9% or less or about 10% or less of the prokaryotic cells in the mixture are lysed into a soluble state.

[0060] Suitable methods and techniques for preferentially lysing mammalian cells in the mixture are known in the art and will be recognised by the skilled person to be useful in the present method. Examples include, without limitation: mild lysis reagents such as detergents and / or surfactants; lysis agents that preferentially target or affect mammalian cells compared to prokaryotic cells; and high salt concentrations. Lysis reagents which may be considered ‘harsh’ or ‘strong’ and which lyse prokaryotic cells (i.e. bacterial cells) are not suitable in the method described herein. In one embodiment, preferential lysis in step (ii) is performed using a detergent and / or surfactant. In a further embodiment, preferential lysis is performed using a toxin. In a yet further embodiment, preferential lysis is performed using high salt ORI-C-P3671 PCT concentrations, such as between 1.5M and 5M final concentration. In a still further embodiment, an ionic and / or non-ionic detergent is used.

[0061] In some embodiments, the detergent and / or surfactant is selected from any one or more of: saponin; sodium dodecyl sulphate (SDS); sodium deoxycholate; sodium cholate; sodium lauroyl sarcosinate; a polyethylene-based detergent such as Triton X-100, Triton X-114, Nonidet P-40 (NP-40), IGEPAL CA-630 and / or Brij-35; a glycosidic surfactant such as n-dodecyl-p-D-maltoside (DDM) and / or digitonin; a polysorbate surfactant such as Tween-20 and / or Tween-80; a glyco-lithocholate amphiphile or glyco-diosgenin amphiphile (GDN); a zwitterionic detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) and / or CHAPSO; and / or a chaotropic agent such as urea and / or thiourea. In a certain embodiment, the detergent and / or surfactant is saponin.

[0062] In further embodiments, the toxin is selected from any one or more of: a streptolysin such as streptolysin O and / or streptolysin S; ricin; abrin; Shiga toxin; and / or diphtheria toxin.

[0063] The method described herein comprises step (iii) of separating the lysed soluble cellular material from the non-lysed insoluble prokaryotic cells. As will be appreciated, lysed cellular material (i.e. preferentially mammalian cellular material) becomes soluble such that it can be transferred / removed in solution from an insoluble fraction. By contrast, non-lysed cells (preferentially prokaryotic cells) remain insoluble and can be separated from a soluble fraction, for example by centrifugation and pelleting.

[0064] Thus, in some embodiments separating in step (iii) comprises pelleting the non-lysed insoluble prokaryotic cells and removing lysed soluble cellular material.

[0065] In alternative embodiments, separating step (iii) comprises binding the lysed soluble cellular material to a solid support, such as magnetic beads. The use of solid supports and beads to bind cellular material is well known in the art and includes in particular paramagnetic beads which bind nucleic acids in the lysed soluble fraction, such as solid-phase reversible immobilization (SPRI) paramagnetic beads. Other solid supports include agarose (e.g. Sepharose). Selection of a suitable solid support / bead material will be apparent to the skilled person depending on the cellular material of interest. For example, wherein the lysed soluble cellular material of interest is nucleic acid, SPRI paramagnetic beads may be used, or wherein the lysed soluble cellular material of interest in protein, Sepharose, optionally Sepharose beads, may be used. Thus, in one embodiment separating step (iii) comprises binding the lysed soluble cellular material to agarose, such as an agarose gel (e.g. in a column) or agarose ORI-C-P3671 PCT beads. In a further embodiment, the beads are Sepharose beads. According to these embodiments, the lysed cellular material of interest is cellular protein. In another embodiment, separating step (iii) comprises binding the lysed soluble cellular material to paramagnetic beads, such as SPRI beads. In a further embodiment, the lysed soluble cellular material may be bound to nitrocellulose or a dextran, such as in the form of a gel in a column or as beads. According to these embodiments wherein paramagnetic beads or nitrocellulose is used, the lysed cellular material of interest is cellular nucleic acid and / or genetic material. In a yet further embodiment, separating step (iii) comprises a capillary flow process, such as capillary hydrodynamic flow fractionation (CHDF), liquid-liquid phase separation (Capflex) and / or capillary electrophoresis.

[0066] Solid Phase Reversible Immobilization (SPRI) beads are known in the art and are common in several commercial and non-commercial kits / protocols for purifying nucleic acids, such as from PCR reaction mixtures. They are paramagnetic (i.e. they are only magnetic when subjected to a magnetic field) which prevents clumping and precipitation during storage and use, and are made of polystyrene surrounded by a layer of magnetite coated with carboxyl molecules. The surface coating of carboxyl molecules reversibly binds to nucleic acids such as DNA in the presence of a crowding agent, such as polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) and a salt, in the binding buffer, which causes the negatively charged nucleic acid molecules to bind to the carboxyl groups, immobilising the nucleic acid molecules on the bead surface. Following immobilisation / binding of nucleic acid molecules on the SPRI beads, a magnetic field is applied to the sample to immobilise the bead / nucleic acid complexes (also known as microparticles) and separate them from the remaining sample mixture. Following one or more wash in the presence of the magnetic field (e.g. using ethanol), the magnetic field is removed and an aqueous elution buffer is added to elute the nucleic acid molecules off the beads and into solution. SPRI beads are easy to handle and their binding capacity for nucleic acids is very large.

[0067] Wherein separating step (iii) comprises binding the lysed soluble cellular material to paramagnetic beads, i.e. wherein the solid support is magnetic, in one embodiment separating comprises subjecting the sample to a magnetic field and removing the non-lysed insoluble prokaryotic cells. According to this embodiment, the lysed soluble cellular material is bound to the magnetic beads and is thus immobilised in the magnetic field, while the non-lysed insoluble prokaryotic cells do not bind to the beads and can be removed and / or washed away. Following removal of the unbound non-lysed prokaryotic cells, bound lysed soluble cellular material is eluted from the beads according to methods well known in the art. Thus, in a further embodiment following removal of the non-lysed insoluble prokaryotic cells, the lysed soluble ORI-C-P3671 PCT cellular material is released from the solid support. In embodiments wherein a solid support in the form of a column is used, release of the lysed soluble material may comprise washing the column with an elution solution to elute the previously bound cellular material. In embodiments wherein the solid support is beads, release of the lysed soluble cellular material may comprise adding an elution solution, pelleting the beads and removing the eluted cellular material in the supernatant. In embodiments wherein the solid support is magnetic beads, release of the lysed soluble material may comprise pelleting and removal of the supernatant as hereinbefore or adding an elution solution, subjecting the beads to a magnetic field to immobilise the beads and removing the eluted cellular material in the supernatant.

[0068] The method described herein comprises step (iv) of removing any prokaryotic cellular material in the lysed soluble cellular material by negative selection using one or more prokaryotic- specific marker. In alternative embodiments, step (iv) comprises removing any prokaryotic cellular material from the lysed soluble cellular material by positive selection using one or more prokaryotic-specific marker as described hereinbefore. The term “removed” herein refers to a separation of any prokaryotic cellular material that may be present in the lysed soluble cellular material following preferential lysis step (ii). As will be appreciated, such prokaryotic cellular material will be soluble but not because of any lysis of prokaryotic cells in step (ii) since such lysis preferentially affects mammalian cells as described hereinbefore, although minimal lysis of prokaryotic cells may occur in step (ii). Therefore, it is believed that such prokaryotic cellular material may be present in the lysed soluble cellular material due to lysis of prokaryotic cells prior to or during sample collection, during sample transport or during handling prior to step (ii). In particular embodiments wherein the mucosal sample is a colorectal mucosal sample, the inventors have found that such lysis of prokaryotic cells is common and can otherwise contaminate a sample of mammalian cellular material if no removing step (iv) is performed. Without being bound by theory, it is believed that prokaryotic cells comprised in colorectal mucosal samples are more susceptible to lysis during sample collection or may already be lysed in the mucosal sample prior to collection, e.g. because the sample is taken low in the gastrointestinal tract by which time certain prokaryotes may be lysed / degraded. This may be in contrast to other sample types or samples collected from mucosal surfaces other than colorectal samples and methods known in the art which are optimised for said samples. For example, in such other samples the prokaryotes may be less susceptible to lysis or remain intact prior to and during sample collection. Also for example, in other methods optimised for other sample types, the prokaryotes may remain intact during sample transport, handling and processing, meaning that removing step (iv) is not required. A particular example is the method described in Charalampous et al. (2019) which does not comprise a removal step (iv). ORI-C-P3671 PCT

[0069] Removing step (iv) herein comprises using one or more prokaryotic-specific marker to specifically ‘pull-out’ prokaryotic cellular material in the lysed soluble cellular material. As will be readily appreciated, such a prokaryotic-specific marker is specific for prokaryotes / is found only in the prokaryotic cellular material, and is not present in the mammalian cellular material. As such, it specifically selects the prokaryotic cellular material either by negative selection and removal for preparing a sample of mammalian cellular material substantially free of prokaryotic cells and / or cellular material, or by positive selection and removal for preparing a sample of prokaryotic cellular material substantially free of mammalian cellular material.

[0070] Examples of prokaryotic-specific markers include any sequence (protein or nucleic acid), protein, cellular component or compound that is found in prokaryotes but not mammalian cells / cellular material. In some embodiments, the one or more prokaryotic-specific marker is prokaryotic genetic material. According to these embodiments, the prokaryotic cellular material in the lysed soluble cellular material in step (iv) therefore comprises prokaryotic genetic material. In one embodiment, the prokaryotic genetic material is prokaryotic DNA. In a further particular embodiment, the prokaryotic genetic material is bacterial DNA. In a yet further embodiment, the prokaryotic genetic material is prokaryotic (e.g. bacterial) RNA. In some embodiments, the one or more prokaryotic-specific marker used in step (iv) is a prokaryotic-specific sequence, such as a genetic sequence of a bacteria-specific gene. In further embodiments, the one or more prokaryotic-specific marker used in step (iv) is bacterial- specific methylation of DNA. Bacterial DNA methylation occurs at adenine and cytosine residues by methylase enzymes which recognise a specific sequence and methylate a base at or near the recognised sequence. Bacteria use this mechanism to recognise ‘self’ from ‘non-self’ DNA (e.g. introduced by a bacteriophage), with non-methylated DNA degraded by restriction enzymes. Adenine methylation is also used during replication to allow DNA repair machinery to differentiate between the template and nascent strands. The methylation of cytosine residues also occurs in mammalian cells. Thus, in one embodiment the bacterial- specific DNA methylation is DNA adenine methylation. In a particular embodiment, the bacterial-specific DNA methylation is N6-methyladenine (N6-m6A). In another embodiment, the bacterial-specific marker is the DNA sequence GmeATC, wherein “meA” is N6-m6A.

[0071] Methods and techniques for recognising bacterial-specific DNA methylation are known in the art and include specific and / or selective binders, including antibodies and other DNA-binding proteins. Thus, some embodiments the bacterial-specific DNA methylation is bound in step (iv) using a bacterial methylation binding protein. Such proteins include methyltransferase enzymes that recognise and bind methylated DNA, such as those belonging to restriction-modification systems and solitary / orphan methyltransferases. ORI-C-P3671 PCT

[0072] Restriction-modification methyltransferases are known for their role in protecting bacteria from invading DNA, methylating ‘self’ DNA and protecting it from their associated restriction endonuclease enzyme (by contrast ‘non-self’ DNA is not methylated and thus degraded by the restriction enzyme). As such, methylation binding proteins from restriction-modification systems useful in the method herein are used alone and without their associated restriction nuclease, as such enzymes will degrade the DNA and not allow for ‘pulling-out’ of the prokaryotic DNA as described hereinbefore. Solitary / orphan methyltransferase enzymes are methyltransferases without an associated restriction nuclease as in restriction-modification systems. They include the DNA adenine methyltransferase (Dam), cell cycle-regulated DNA MTase (Ccrm) and DNA cytosine methyltransferase (Dem) enzymes. Dam is a 32 kDa monomeric protein that catalyzes the transfer of the methyl group from AdoMet to the N6 position of the adenine residue in 5'GATC3' sequences. Dam has similar efficiency for both hemi- and unmethylated templates and has been shown to be involved in chromosome replication and segregation, DNA mismatch repair, regulation of transposition events, phase variation and bacterial conjugation processes (Sanchez-Romero et al. (2015) Curr. Opin.

[0073] Microbiol. 25:9-16, doi: https : / / doi, prg / 10, 10 S / j, m sb.20 5,03.004) . CcrM is a functional monomer but may form a dimer at physiological concentrations (Shier et al. (2001) J. Biol. represents any base) site can be methylated and modified before the enzyme detaches from the DNA (Marinus and Casadesus (2009) FEMS Microbiol. Rev., 33(3):488-503, doi: Dem is mainly found in enterobacteria such as E. coli and is a 51 KD protein that methylates cytosine in a position that is rarely modified in bacteria but commonly in eukaryotes. As a C5-cytosine methyltransferase, Dem can methylate the second cytosine in 5'CCAGG 3' and 5'CCTGG 3' sites (Militello et al. (2014) FEMS Microbiol. Lett., 350(1):100-106, doi: https: / / dQs.Qrq / 10. 111 / 874-0988. 2209 ). As a cytosine methyltransferase which thus also recognises methylated residues in eukaryotic (i.e. mammalian) DNA, it is not suitable for use in the present method. Thus, in one embodiment the bacterial methylation binding protein is a methyltransferase selected from: Dam and / or Ccrm. In a particular embodiment, the bacterial methyltransferase is Dam. ORI-C-P3671 PCT

[0074] To facilitate the removal in step (iv) of the prokaryotic DNA (e.g. bacterial DNA) from the lysed soluble cellular material, wherein a bacterial methylation binding protein is used it may be conjugated to a further protein or tag. Such conjugated protein / tag can be ‘pulled-out’ and / or precipitated, such as immunoprecipitated, together with the methylation binding protein and bound prokaryotic DNA. In some embodiments, the methylation binding protein is conjugated to protein A, protein G, protein A / G and / or protein L. Protein A, protein G, protein A / G and protein L are bacterial proteins that bind to the Fab and Fc region of immunoglobulins, i.e. antibodies. Protein A and protein G are expressed in streptococcal bacteria, protein L is from the bacterial species Peptostreptococcus magnus and protein A / G is a recombinant fusion protein of the binding domains of protein A and protein G. According to these embodiments, antibodies may subsequently be used to remove the conjugated methylation binding protein, together with bound prokaryotic DNA, from the lysed soluble cellular material.

[0075] Alternatively, the methylation binding protein may be recognised by an anti-methyltransferase antibody. Suitable antibodies that are specific and / or selective for bacterial methyltransferases and other bacterial methylation binding proteins are known in the art. Thus, in other embodiments the bacterial methylation binding protein is bound in step (iv) using an antibody. In one embodiment, the antibody is an anti-methyltransferase antibody. In a further particular embodiment, the antibody is an anti-Dam antibody.

[0076] In other embodiments, the bacterial-specific DNA methylation is bound in step (iv) using an antibody, i.e. the DNA methylation is directly bound using an antibody. Suitable antibodies that are specific and / or selective for prokaryotic / bacterial-specific DNA methylation are known in the art. As described hereinbefore, DNA adenine methylation, in particular N6-m6A, is specific to bacteria. Thus, in one embodiment the antibody is an anti-methyl adenine antibody. In a further particular embodiment, the antibody is an anti-N6-m6A antibody.

[0077] In further embodiments, the methylation binding protein and / or antibody may be immobilised. Such immobilisation may be on a solid support such as in the form of beads or a column as described hereinbefore. Thus, in one embodiment the methylation binding protein and / or antibody may be immobilised on agarose, such as an agarose gel (e.g. in a column) or agarose beads. In a further embodiment, the beads are Sepharose beads. In a yet further particular embodiment, the beads may be magnetic, such as paramagnetic.

[0078] In further embodiments, the methylated bacterial DNA is removed in step (iv) by enzymatic digestion using a methylation-specific or methylation-dependent restriction enzyme. ORI-C-P3671 PCT

[0079] Methylation-dependent restriction enzymes require their recognition site to be methylated in order for cleavage to occur. Thus, according to these embodiments methylated bacterial DNA will be preferentially digested compared to mammalian genetic material in the lysed soluble cellular material. Examples of methylation-specific or methylation-dependent restriction enzymes are known in the art and include, without limitation, Dpnl, Dpnll, Mspl, Hpall, MrcBC, as well as MspJI, FspEI, LpnPI, AspBHI and other Mrr-like and type IV restriction enzymes. Thus, in a particular embodiment the methylation-specific / dependent restriction enzyme is Dpnl. In further embodiments, the methylation-specific / dependent restriction enzyme is an Mrr-like and / or a type IV restriction enzyme.

[0080] As described hereinbefore, the present method finds utility in preparing / separating any mammalian cellular material, including proteins. Thus, in some embodiments the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic protein, such as bacterial protein. According to these embodiments, the one or more prokaryotic- specific marker is one or more prokaryotic protein, such as one or more bacterial protein. In a particular embodiment, the one or more prokaryotic-specific marker used in step (iv) is one or more bacterial-specific protein. Suitable bacterial-specific proteins are known in the art, and include for example a bacterial methyltransferase as described hereinbefore. In a further embodiment, the one or more bacterial-specific protein is bound in step (iv) using one or more antibody. However, as will be readily appreciated, wherein the one or more prokaryotic- specific marker is one or more prokaryotic protein, a panel of multiple proteins will be used in the present method due to the need to remove substantially all prokaryotic cellular material (i.e. substantially all prokaryotic proteins according to these embodiments). This is in contrast to wherein bacterial-specific DNA methylation is used which is present throughout the bacterial genome and a single binding protein / antibody can be used to remove substantially all prokaryotic genetic material. Thus, in a yet further embodiment the one or more bacterial- specific protein is bound in step (iv) using a panel of (i.e. multiple) anti-bacterial antibodies. As described hereinbefore, the antibody / antibodies may be immobilised on a solid support, such as in the form of beads or a column (e.g. magnetic / paramagnetic beads).

[0081] Following binding of the one or more prokaryotic-specific markers, step (iv) comprises separating the bound prokaryotic cellular material from the unbound mammalian cellular material. Thus, in one embodiment step (iv) comprises removing the unbound mammalian cellular material from the bound prokaryotic cellular material. Said removal / separation will depend on the method used to bind the prokaryotic cellular material using the prokaryotic- specific marker and may use any suitable method / technique known in the art. For example, wherein one or more antibodies are used, removing may be by immunoprecipitation of the ORI-C-P3671 PCT antibodies and prokaryotic cellular material bound thereto. Immunoprecipitation may also be used when antibodies are used to bind a protein A, protein G, protein A / G and / or protein L conjugated methylation binding protein. Thus, in a further embodiment removing is by immunoprecipitation of the bound prokaryotic cellular material.

[0082] As will be readily appreciated, any of the hereinbefore described methods suitable for removing the prokaryotic cellular material in the lysed soluble cellular material in step (iv) may be used individually or together, such as sequentially or in combination simultaneously. Thus, in some embodiments step (iv) herein may be repeated one or more further times.

[0083] Kits

[0084] According to another aspect of the invention, there is provided a kit for preparing a sample of mammalian cellular material from a mucosal sample, which comprises buffers and reagents capable of performing the method described herein. Thus, in embodiments the kit yields a sample of mammalian cellular material substantially free of prokaryotic cells and / or prokaryotic cellular material.

[0085] The kit may include one or more articles and / or reagents for performance of the method. For example, a preferential lysis solution and reagents for recognising a prokaryotic-specific marker for use in the method described herein may be provided in isolated form and may be part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment. Further provided may be reagents suitable for analysing the sample of mammalian cellular material as described herein. Such further reagents for analysis may include adapter sequences for sequencing, or primers for PCR. The kit may include instructions for use according to the protocol of the method described herein.

[0086] Uses, Diagnosis & Treatment of Disease

[0087] As described hereinbefore, the method may further comprise analysing the sample of mammalian cellular material. Such analysis may be used to detect the presence of absence of a marker associated with disease, with said marker being causative or otherwise directly involved in disease or being a biomarker. For example, wherein the disease is associated with a mutation, the mammalian cellular material may be genetic material and its sequence analysed. In another example, wherein the disease is associated with an increased level or activity of a protein, the mammalian cellular material may be protein and the presence, level and activity of the protein associated with disease analysed. Thus, in further embodiments the sample is analysed to detect the presence or absence of a marker associated with a disease or disorder. ORI-C-P3671 PCT

[0088] In a particular embodiment, the mammalian cellular material is genetic material and the disease / disorder-associated marker is a genetic change associated with disease ora disorder. Well known examples of such diseases include various cancers. Thus, as will be appreciated, the method described herein finds utility in analysing samples for the presence or likelihood of cancers of mucosa, such as epithelial cancer and in particular colorectal cancer and endometrial cancer. In particular embodiments, the disease or disorder is cancer. In a further embodiment, the disease or disorder is a cancer of a mucosal surface. In a yet further embodiment, the cancer is epithelial cancer. In a still further embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is endometrial cancer. In other embodiments, the disease or disorder is a non-cancerous condition of the gastrointestinal tract (Gl). Examples of Gl conditions include, without limitation, inflammatory bowel disease (IBD), diverticular disease and Crohn’s disease. In still other embodiments, the disease or disorder is a gynaecological disease / disorder, for example and without limitation, ovarian cancer endometriosis and pre-eclampsia uterine cancer.

[0089] In further embodiments, the genetic change associated with disease or a disorder is a single nucleotide polymorphism (SNP), a mutation and / or a change in gene expression. As known in the art, such SNPs, mutations and gene expression changes can be correlated with various diseases, including cancer. Methods of identifying suitable SNPs for the purpose of determining disease are well known in the art. In embodiments where a change in gene expression is analysed, the mammalian cellular material may be genetic material, in particular RNA, and / or protein, both of which provide information as to the level of gene expression. Thus, in one embodiment the mammalian cellular material may be non-genetic material (e.g. protein) and the disease / disorder associated marker is a change in the level and / or activity associated with disease or a disorder. In a further embodiment, the mammalian cellular material is protein and the level and / or activity of the protein is altered in disease. Said protein level and / or activity may be increased in disease or decreased. In a yet further embodiment, the mammalian cellular material may be both genetic material and protein, and the disease / disorder-associated marker is a genetic change and a change in protein level and / or activity associated with disease or a disorder.

[0090] The identification and / or analysis of markers associated with disease and disorders finds utility in diagnosing said disease / disorder in a subject. Diagnosis includes determining the likelihood that the subject has / is suffering from the disease / disorder or will develop the disease / disorder. ORI-C-P3671 PCT

[0091] Thus, in a further aspect of the present invention there is provided a method of diagnosing a disease or disorder in a mammalian subject, the method comprising obtaining a sample of mammalian cellular material according to the method described herein and analysing said sample for a marker associated with the disease or disorder. In one embodiment, the presence of the marker indicates a likelihood that the subject is suffering from or has the disease or disorder. In a further embodiment, the absence of the marker indicates that the subject is unlikely to be suffering from or has the disease or disorder. Such presence and absence may be combined across multiple markers, for example with oncogenes and tumoursuppressor genes. In alternative embodiments, the presence of the marker indicates that the subject is unlikely to be suffering from or has the disease or disorder and / or the absence of the marker indicates that the subject is likely to be suffering from or has the disease or disorder.

[0092] Following diagnosis, the identified disease or disorder can be treated using a suitable treatment regime and / or medicament for said disease / disorder. Thus, in a yet further aspect there is provided a method of treating a disease or disorder in a mammalian subject in need thereof, said method comprising the steps of:

[0093] (i) performing the method of analysis diagnosis described herein on a mucosal sample obtained from the subject; and

[0094] (ii) administering to the subject a treatment for the disease or disorder if the disease / disorder-associated marker is present.

[0095] As will be readily appreciated, such treatment may be administered based on the presence of the disease / disorder-associated marker, its absence or a combination as described hereinbefore.

[0096] It will be appreciated that references herein to a patient or subject relate equally to animals and humans and that the invention finds particular utility in veterinary treatment of any of the above mentioned diseases, disorders and conditions which are also present in said animals.

[0097] It will also be appreciated that references herein to “treatment” and “amelioration” include such terms as “prevention”, “reversal” and “suppression”. Similarly, references to “diagnosis” and “identification of disease state” may be used interchangeably herein and include terms such as “differential diagnosis”, “patient stratification” and the like. Furthermore, such terms include reference to the “patient”, “subject” and “individual” (e.g. an individual / subject in need of treatment) which may be used interchangeably herein. Treatment and / or diagnosis may be prior to the onset of the disease or disorder, e.g. wherein the subject is at risk of the disease ORI-C-P3671 PCT or disorder. Alternatively, treatment and / or diagnosis may also be anticipated after the induction event of the injury, damage, disease or disorder, either before clinical presentation of said disease or disorder, or after symptoms manifest. Such references further include performing the methods of analysing the mammalian cellular material either prior to the onset of the disease, or after the induction event of the disease.

[0098] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the term “about” when used herein includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater and up to and including 5% lower than the value specified, especially the value specified. The term “between” as used herein includes the values of the specified boundaries.

[0099] Throughout the specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations thereof such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

[0100] In addition, as used herein and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise (e.g. in a context where the term “single” is clearly required). Thus, for example reference to “a prokaryotic-specific marker” includes two or more such markers, and reference to “an antibody” includes two or more antibodies, such as a panel comprising multiple antibodies.

[0101] It will be understood that all embodiments described herein may be applied to all aspects of the invention and vice versa.

[0102] Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples:

[0103] EXAMPLES

[0104] Example 1: Preferential Lysis

[0105] An exemplary protocol for performing the preferential lysis steps of the method of the invention is as follows: ORI-C-P3671 PCT

[0106] 1 . Aliquot 250|JL of colorectal mucosal sample in stabilisation buffer, into a 1.5 mL tube.

[0107] 2. Add 2.2% Saponin in a total volume of 300pL.

[0108] 3. Incubate at room temperature for 10 minutes, with periodic gentle shaking.

[0109] 4. Add 245pL of 5M NaCh and mix by inversion.

[0110] 5. Incubate the sample at 37°C for 15mins, whilst shaking at 500rpm.

[0111] 6. Add SPRI beads at a 1.8:1 ratio.

[0112] 7. Mix at 800rpm for 5 minutes at room temperature.

[0113] 8. Beads are bound to a magnetic and supernatant is removed into a separate tube (tube 1 , microbial fraction).

[0114] 9. The bottom of the sample tube is rinsed in 300pL PBS and transferred to tube 1.

[0115] 10. The beads are then resuspended in 50pL elution buffer and transferred to a separate tube (tube 2).

[0116] 11 . The human fraction is then released from the beads (in tube 2) by mixing at 800rpm and heating at 65°C for 5 minutes.

[0117] The isolated human fraction (tube 2) contains a significant quantity of bacterial DNA (though heavily reduced compared to in the starting material), as a proportion of the bacterial cells are lysed upon sample isolation. The method now forks, with various additional procedures that are capable of further enrichment.

[0118] Example 2: Antibody Mediated Pulldown of Bacterial DNA / Associated Binding Proteins (Method 1)

[0119] An exemplary protocol for the step(s) of removing prokaryotic cellular material of the invention is as follows:

[0120] 1. Prepare 0.05% PBS / tween solution by adding 5pL Tween 20 to 10mL PBS.

[0121] 2. Each sample requires 1 ,5pg of each antibody conjugated to separate 50pL Pierce protein A beads each (500pg as protein A beads are supplied at 10mg / mL).

[0122] 3. Conjugate each antibody separately as follows, include 10% overage when calculating requirements.

[0123] 4. Aliquot necessary volume of protein A beads (number of samples x 50pL( / 500pg) protein A beads).

[0124] 5. Wash beads with 500pl 0.05% PBS / Tween 20, gently pipette mix.

[0125] 6. Place the tube on magnetic rack for 2 mins or until the solution is clear, discard the supernatant.

[0126] 7. Repeat step 6.

[0127] 8. Resuspend the beads in 2mL minus the volume of antibody required. ORI-C-P3671 PCT

[0128] 9. Add volume of antibody required (number of samples x cone. - e.g. either 0.5ng / pL for RB1 or 1ng / pL for m6A).

[0129] 10. Pipette mix and gently vortex. Incubate for >1 hr at RT and 600-800rpm.

[0130] 11 . Remove from bead / antibody mix from the shaker.

[0131] 12. Place the tube on magnetic rack for 2 mins or until the solution is clear, discard the supernatant.

[0132] 13. Wash beads with 500pl 0.05% PBS / Tween 20, gently pipette mix.

[0133] 14. Place the tube on magnetic rack for 2 mins or until the solution is clear, discard the supernatant.

[0134] 15. Repeat step 14.

[0135] 16. Resuspend beads in required volume of 0.05% PBS / Tween 20 (number of samples x 50pL), pipette mix and gently vortex.

[0136] 17. Per sample, add 50pL of protein A beads conjugated to M6A antibody and 50pL of protein A beads conjugated to RB1 antibody, to previously collected human fraction.

[0137] 18. Incubate at RT overnight with gentle shaking (800 rpm).

[0138] 19. Ensure SPRI beads have equilibrated to room temperature before use.

[0139] 20. Place incubated samples on magnet rack and transfer the supernatant (~300pL) as the human fraction to a fresh 1 .5 mL tube (-AbSup).

[0140] 21. Add 540pL SPRI bead suspension to 300pL human fraction Sample (1.8x).

[0141] 22. Mix thoroughly, incubate 5 mins RT.

[0142] 23. Place the tube on magnetic rack for 2 mins or until the solution is clear, discard the supernatant.

[0143] 24. While keeping the sample on the magnet, wash with 800pL 80% EtOH, incubate for 30s and discard supernatant.

[0144] 25. Repeat step 24.

[0145] 26. Add 100pL NF-H2O and fully resuspend SPRI beads by pipette mixing.

[0146] 27. Incubate for 5 min at 70°C in a sample shaker at 800 rpm.

[0147] 28. Place the tube on magnetic rack for 2 mins or until the solution is clear.

[0148] 29. Transfer supernatant to a fresh 96-well plate. These constitute the final purified samples.

[0149] As shown in Figure 1 , method 1 is able to increase the purity of the human fraction by approx. 10-15%, with 70-90% of total bacterial mass removed in the positive fraction (bacterial DNA / associated binding proteins-antibody complexes). As demonstrated in Example 5, an increase in purity of 10-15% as achieved by method 1 is sufficient to greatly increase the likelihood of a sample meeting the required 98% conversion rate for methylome assessment. ORI-C-P3671 PCT

[0150] Example 3: Specific Endonuclease Digestion of Bacterial DNA and Size Specific Purification (Method 2)

[0151] A further exemplary protocol for the step(s) of removing prokaryotic cellular material of the invention is as follows:

[0152] 1. Prepare the Dpnl digestion mastermix as below per reaction:

[0153] 2. Add the relevant volume of DNA (up to 1 pg total mass) and add H20 to a total volume of 100pl.

[0154] 3. Incubate each sample at 37°C for 4 hr, followed by 20 min at 80°C.

[0155] 4. Remove sample from the heat block / thermocycler and allow to cool to RT.

[0156] 5. Add 50pl of SPRI beads to each completed reaction, mix by pipetting and for 1 min at 2000rpm on a plate shaker.

[0157] 6. Incubate each reaction for 4 mins at RT, with periodic vortex mixing.

[0158] 7. Apply sample bead mixes to magnetic stand / plate for 2 mins or until the solution is clear.

[0159] 8. Discard supernatant.

[0160] 9. Whilst on the magnet add 200pl of 80% ETOH to each sample, incubate at RT for 30s, before discarding the supernatant.

[0161] 10. Repeat step 9.

[0162] 11. Carefully remove all remaining ETOH residue from each sample and allow the beads to dry for 1 min or until the beads have no residue. Do no allow the beads to over dry.

[0163] 12. Add 30ul of H20 to each sample, resuspend the beads by pipette mixing, followed by 1 min at 2000 RPM on a plate shaker.

[0164] 13. Incubate samples for 4 mins at RT, with periodic vortex mixing.

[0165] 14. Apply sample bead mixes to magnetic stand / plate for 2 mins or until the solution is clear.

[0166] 15. Transfer supernatant to a fresh 1.5mL tube / 96-well plate. These constitute the final purified samples.

[0167] As shown in Figure 2, method 2 is able to increase the purity of the human fraction by up to 30% (i.e. undigested non-bacterial DNA). As demonstrated in Example 5, an increase in purity of approx. 30% as achieved by method 2 is sufficient to greatly increase the likelihood of a sample meeting the required 98% conversion rate for methylome assessment. ORI-C-P3671 PCT

[0168] Example 4: Negative Selection of Human DNA by Hybridisation Capture of Generic Bacterial Regions (Method 3)

[0169] A further exemplary protocol for the step(s) of removing prokaryotic cellular material of the invention is as follows:

[0170] 1. Shear human fraction DNA to 250-350 bp.

[0171] 2. Prepare hybridisation mastermix (below):

[0172] 3. Incubate hybridisation mastermix at 60°C for 10 minutes, with periodic vortex mixing.

[0173] 4. For each capture reaction, aliquot 18.5pL of hybridisation mastermix to the well of a 96 well plate (“HYBs”).

[0174] Prepare blocker mastermix (below):

[0175] 5. For each capture reaction, aliquot 5pL of blocker mastermix to the well of a 96 well plate.

[0176] 6. Add 7pL of individual / pooled libraries to each aliquot of blocker mastermix and mix by pipetting (“LIBs”).

[0177] 7. Put the LIBs in the thermal cycler, close lid and start cycling conditions (below):

[0178] 8. Once the cycler reaches hybridisation (1) temperature, pause the program and add the HYBs to a separate well of the 96 well plate. ORI-C-P3671 PCT

[0179] 9. Close the lid and resume the program.

[0180] 10. After hybridization (1) step is complete, pause the program and whilst leaving the 96 well plate in the thermocycler, pipette 18pl of each HYB to each LIB. Mix by pipetting up and down 5 times.

[0181] 11 . Briefly spin down the 96 well plate before returning to the thermocycler and resuming the program.

[0182] 12. One hour before the program is completed prepare wash buffers and capture beads.

[0183] 13. Wash buffer preparations are completed as below, each reaction is adequate for the 4 required washes per reaction:

[0184] 14. For each capture reaction aliquot 30pL of capture beads.

[0185] 15. Pellet beads by centrifugation and discard supernatant.

[0186] 16. Resuspend capture beads in 200pL of binding buffer per capture reaction.

[0187] 17. Pellet beads by centrifugation and discard supernatant.

[0188] 18. Repeat steps 14 to 15 an additional two times.

[0189] 19. Resuspend washed capture beads in 70pL per reaction.

[0190] 20. Heat washed beads to 60°C for 2 mins.

[0191] 21. Transfer LIBs to beads and mix by pipetting and brief vortex.

[0192] 22. Incubate bead / LIB mixes at 60°C for 5 min, with a vortex mix and push centrifugation step at 2.5 min.

[0193] 23. Pellet beads by pulse centrifugation and apply sample plate to 96-well plate magnet for 2 mins or until the solution is clear.

[0194] 24. Transfer the supernatant (90pL) to a new 96-well plate (NEG FRACTION).

[0195] 25. Add 162pL of SPRI beads to the NEG FRACTION, mix by pipetting and for 1 min at 2000rpm on plate shaker.

[0196] 26. Incubate Negative Fraction 4 mins at RT, with periodic vortex mixing.

[0197] 27. Apply NEG FRACTION plate to 96-well pate magnet for 2 mins or until the solution is clear.

[0198] 28. Discard supernatant.

[0199] 29. With the plate still on the magnet add 200pL of 80% ETOH to each NEG FRACTION, incubate at RT for 30s, before discarding the supernatant.

[0200] 30. Repeat step 29. ORI-C-P3671 PCT

[0201] 31. Carefully remove all remaining ETOH residue from NEG FRACTION samples and allow the beads to dry for 1 min or until the beads have no residue. Do no allow the beads to over dry.

[0202] 32. Add 30pl of H20 to each NEG FRACTION sample, resuspend the beads by pipette mixing, followed by 1 min at 2000rpm on a plate shaker.

[0203] 33. Incubate Negative Fraction 4 mins at RT, with periodic vortex mixing.

[0204] 34. Apply NEG FRACTION plate to 96-well pate magnet for 2 mins or until the solution is clear.

[0205] 35. Transfer supernatant to a fresh 96-well plate. These constitute the final purified samples.

[0206] As shown in Figure 3, method 3 using the targeted removal of representative conserved regions of the bacterial genome, is able to significantly increase the purity of the human cellular material mass and reduce the amount of bacterial cellular mass, compared to bulk extraction methods.

[0207] Example 5: Analysis of Purified Human DNA

[0208] It is vitally important to remove as much of the bacterial DNA as possible from a sample (in particular a colorectal mucosal sample as described herein), when downstream applications are intended to analysis the human proportion of the sample. Increased bacterial load can result in a litany of failures in conventional molecular biological techniques, for example:

[0209] Hybridisation / capture biases

[0210] Enzymatic inhibition

[0211] Off-target sequencing

[0212] Preferential sequencing of undesirable contaminates (microbes)

[0213] A particular example is the enzymatic inhibition / quenching seen during enzymatic conversion, a key process in the assessment of methylation epigenetic markers. The conversion is a crucial step of methylome assessment, with CpG conversion required to be above 98%, to reliably analyse the data. Percentage conversion rate is measured by the number of non-CpG cytosines converted in the sample and acts as a representative, simple to assess metric for the efficiency of the process and quality control metric.

[0214] Figure 4 details the non-CpG conversion rate (labelled “CpG”) from multiple samples extracted in a conventional manner, compared to those enriched by method 1 and method 2, as detailed above. In order for samples to be reliably analysable, the conversion rate must be above 98%. The 10 samples assessed all failed to meet this criteria, without enhanced ORI-C-P3671 PCT enrichment, but following enrichment using either method 1 or method 2, 8 out of the 10 samples achieved conversion rates above 98%.

[0215] Example 6: Extended Analysis of Purified Human DNA

[0216] Further investigation into sample purity dependent methylome detection was conducted using a larger methylome targeting panel (Human Methylome Panel, Twist Bioscience) in combination with the aforementioned enhanced enrichment methods. This assessment was conducted in in an augmented clinical cohort of collected rectal mucus samples (n = 496), to ascertain whether the illustrated findings remained consistent across a large heterogeneous clinical cohort.

[0217] The conversion is a crucial step of methylome assessment, with CpG conversion required to be above 98%, to reliably analyse the data. Percentage conversion rate is measured by the number of non-CpG cytosines converted in the sample and acts as a representative, simple to assess, metric for the efficiency of the process and quality control metric.

[0218] Tables 1-7 detail the human non-CpG conversion rate (labelled “Human_nonCpG_conversion_rate”) from six clinical sample batches. The samples processed utilised method 2, as detailed above. In order for samples to be reliably analysable, the conversion rate must be above 98%. The results show the conversion rate for each sample (labelled “Barcode”) by batch, percentage of samples achieving >98% conversion per batch and for the combined cohort. The expanded assessment illustrates that 95.77% of the 496 samples assessed reached this >98% threshold.

[0219] Table 1 ORI-C-P3671 PCT ORI-C-P3671 PCT

[0220] Table 2 ORI-C-P3671 PCT

[0221] Table 3 ORI-C-P3671 PCT ORI-C-P3671 PCT ORI-C-P3671 PCT

[0222] Table 4 ORI-C-P3671 PCT ORI-C-P3671 PCT

[0223] Table 5 ORI-C-P3671 PCT ORI-C-P3671 PCT

[0224] Table 6 ORI-C-P3671 PCT ORI-C-P3671 PCT

[0225] Table 7

Claims

1. ORI-C-P3671 PCTCLAIMS1. A method of preparing mammalian cellular material from a mucosal sample, comprising the steps of:(i) obtaining a mucosal sample comprising a mixture of mammalian and prokaryotic cells;(ii) preferentially lysing the mammalian cells in the mixture into a soluble state while retaining substantially all of the prokaryotic cells in a non-lysed insoluble state;(iii) separating the lysed soluble cellular material from the non-lysed insoluble prokaryotic cells; and(iv) removing any prokaryotic cellular material in the lysed soluble cellular material using one or more prokaryotic-specific marker, thereby yielding a sample of mammalian cellular material with reduced prokaryotic cells and / or prokaryotic cellular material compared to the obtained mucosal sample.

2. The method of claim 1 , additionally comprising the step of:(ib) separating any soluble cellular material from the mucosal sample prior to preferentially lysing the mammalian cells in step (ii), and adding the separated soluble cellular material to the lysed soluble cellular material prior to step (iv).

3. The method of claim 1 or claim 2, wherein the mucosal sample is a colorectal mucosal sample, in particular a human colorectal sample comprising a mixture of human and bacterial cells.

4. The method of any one of claims 1 to 3, wherein the mucosal sample is obtained using a cell sampling device, optionally wherein the mucosal sample is not diluted prior to step (ii), and / or optionally wherein cells in the mucosal sample are not pre-purified prior to step (ii), in particular wherein mammalian cells in the mucosal sample are not pre-purified prior to step (ii).

5. The method of any one of claims 1 to 4, wherein preferential lysis in step (ii) is performed using a detergent and / or surfactant, a toxin and / or high salt concentrations, such as an ionic and / or non-ionic detergent, optionally wherein the detergent and / or surfactant is selected from any one or more of: saponin; sodium dodecyl sulphate (SDS); sodium deoxycholate; sodium cholate; sodium lauroyl sarcosinate; a polyethylene-based detergent such as Triton X-100, Triton X-114,ORI-C-P3671 PCTNonidet P-40 (NP-40), IGEPAL CA 630 and / or Brij-35; a glycosidic surfactant such as n dodecyl-p-D-maltoside (DDM) and / or digitonin; a polysorbate surfactant such as Tween-20 and / or Tween-80; a glyco-lithocholate amphiphile or glyco-diosgenin amphiphile (GDN); a zwitterionic detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS) and / or CHAPSO; and / or a chaotropic agent such as urea and / or thiourea, and / or optionally wherein the toxin is selected from any one or more of: a streptolysin such as streptolysin O and / or streptolysin S; ricin; abrin; Shiga toxin; and / or diphtheria toxin.

6. The method of any one of claims 1 to 5, wherein separating in step (iii) comprises pelleting the non-lysed insoluble prokaryotic cells and removing lysed soluble cellular material.

7. The method of any one of claims 1 to 5, wherein separating in step (iii) comprises binding the lysed soluble cellular material to a solid support, such as magnetic beads.

8. The method of claim 7, wherein the solid support is magnetic and separating comprises subjecting the sample to a magnetic field and removing the non-lysed insoluble prokaryotic cells.

9. The method of claim 7 or claim 8, wherein following removal of the non-lysed insoluble prokaryotic cells, the lysed soluble cellular material is released from the solid support.

10. The method of any one of claims 1 to 9, wherein the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic genetic material, such as prokaryotic DNA, in particular bacterial DNA.11 . The method of any one of claims 1 to 10, wherein the one or more prokaryotic-specific marker used in step (iv) is bacterial-specific methylation of DNA, such as DNA adenine methylation, in particular N6-methyladenine (N6-m6A).

12. The method of claim 11 , wherein the bacterial-specific DNA methylation is bound in step (iv) using a bacterial methylation binding protein, such as a bacterial methyltransferase, in particular DNA adenine methylase (Dam), optionally wherein the bacterial methylation binding protein is conjugated to protein A, protein G, protein A / G and / or protein L.ORI-C-P3671 PCT13. The method of claim 11 or claim 12, wherein the bacterial-specific DNA methylation is bound in step (iv) using an antibody, such as an anti-methyl adenine antibody, in particular an anti-N6-methyladenine (anti-N6-m6A) antibody, or wherein the bacterial methylation binding protein is bound in step (iv) using an antibody, such as an anti-methyltransferase antibody, in particular an anti-Dam antibody.

14. The method of claim 12 or claim 13, wherein the bacterial methylation binding protein and / or the antibody is immobilised, such as on a solid support, in particular on magnetic beads.

15. The method of claim 11 , wherein the methylated bacterial DNA is removed in step (iv) by enzymatic digestion using a methylation-specific or methylation-dependent restriction enzyme, such as Dpnl.

16. The method of any one of claims 1 to 15, wherein the prokaryotic cellular material in the lysed soluble cellular material in step (iv) comprises prokaryotic protein, such as bacterial protein.

17. The method of any one of claims 1 to 16, wherein the one or more prokaryotic-specific marker used in step (iv) is one or more bacterial-specific protein.

18. The method of claim 17, wherein the one or more bacterial-specific protein is bound in step (iv) using one or more antibody, such as a panel of anti-bacterial antibodies, optionally wherein the one or more antibody is immobilised, such as on a solid support, in particular on magnetic beads.

19. The method of any one of claims 12 to 14 or claim 18, wherein step (iv) comprises removing the unbound mammalian cellular material from the bound prokaryotic cellular material, optionally wherein said removing is by immunoprecipitation of the bound prokaryotic cellular material.

20. The method of any one of claims 1 to 19, wherein the method further comprises the step of:(v) analysing the sample of mammalian cellular material, optionally by sequencing wherein the mammalian cellular material is genetic material, such as DNA and / or RNA.ORI-C-P3671 PCT21. The method of claim 20, wherein the sample is analysed to detect the presence or absence of a marker associated with a disease or disorder, optionally wherein the mammalian cellular material is genetic material and the disease / disorder-associated marker is a genetic change associated with disease or a disorder, such as a single nucleotide polymorphism (SNP), a mutation and / or a change in gene expression, and / or optionally wherein the mammalian cellular material is non-genetic material and the disease / disorder associated marker is a change in the level and / or activity associated with disease or a disorder, such as wherein the mammalian cellular material is protein and the level and / or activity of the protein is altered in disease.

22. A method of diagnosing a disease or disorder in a mammalian subject, the method comprising obtaining a sample of mammalian cellular material according to the method of any one of claims 1 to 21 and analysing said sample for a marker associated with the disease or disorder, such that presence of the marker indicates a likelihood that the subject is suffering from the disease or disorder and absence of the marker indicates that the subject is unlikely to be suffering from the disease or disorder.

23. A method of treating a disease or disorder in a mammalian subject in need thereof, said method comprising the steps of:(i) performing the method of claim 21 on a mucosal sample obtained from the subject; and(ii) administering to the subject a treatment for the disease or disorder if the disease / disorder-associated marker is present.

24. The method of any one of claims 21 to 23, wherein the disease or disorder is cancer, such as a cancer of a mucosal surface, in particular an epithelial cancer such as selected from colorectal cancer or endometrial cancer.

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