WO2022212610A1 - Fast, automated method for characterization of nonspecific pathogens in a sample - Google Patents

Abstract

Methods, systems, and apparatus, including computer programs encoded on computer storage media for characterization of nonspecific pathogens in a sample. Provided herein are methods and systems that may be used for the fast, automated detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample. A system may perform the operations of selectively lysing host cells using saponin, depleting host RNA/DNA using propidium monoazide, lysing pathogens using sporeLYSE, extracting pathogen DNA, preparing the pathogen DNA library for sequencing through fragmentation and addition of adapters, sequencing the pathogen DNA library using nanopore sequencing, and analyzing the sequence data.

Description

A FAST, AUTOMATED METHOD FOR CHARACTERIZATION OF NONSPECIFIC

PATHOGENS IN A SAMPLE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 63/169,871 filed on April 1, 2021, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] The present invention relates to methods and systems for the fast, automated detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample.

BACKGROUND

[0003] Rapid detection and characterization of infectious pathogens in clinical samples is a significant challenge, but it is critical to effective diagnostics and therapy. Traditional methods rely on the culturing of clinical samples, a process that can take days or weeks depending on the sample. Additional steps are required if the sample is to be characterized - such as determining its antibiotic resistance - further increasing the turnaround time. Moreover, these methods can miss pathogens that cannot be cultivated by standard techniques. While recent developments in PCR and mass spectrometry-based methods have offered improvements in speed, these are still limited to a narrow range of search targets, thus leaving open the possibility of missing a critical diagnosis. Furthermore, neither of these two approaches incorporate antimicrobial resistance characterization. As such, there exists a high demand for diagnostic methods that can facilitate timely therapy.

[0004] The present application meets these challenges by providing a rapid system and method for the detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample. This approach harnesses a unique reagent composition to lyse nonspecific pathogens - including bacterial and fungal cells - to enable extraction of pathogenetic DNA, which is then sequenced via nanopore sequencing. These sequences are analyzed through cloud-based bioinformatic software, the results of which are returned in a user-friendly format. Aside from the advantages of speed and nonspecificity, the present application also provides simple, point-of-care use without the need to ship samples to a culturing facility, as well as multiplexing to process multiple samples simultaneously.

SUMMARY

[0005] Provided herein are methods and systems for the fast, automated detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample. The disclosed methods comprise: (a) user input of sample; (b) depletion of the host intracellular RNA/DNA; (c) depletion of the host extracellular RNA/DNA; (d) extraction of the pathogen DNA; (e) preparation of the DNA library for sequencing; (f) sequencing; (g) sequence analysis; and (h) delivery of results to the user.

[0006] In some embodiments, the input sample is a biofluid. In some embodiments the input sample includes, but is not limited to blood, saliva, excreta, lymph, perilymph, endolymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, serous fluid, joint fluid, interstitial fluid, and/or transcellular fluid.

[0007] In some embodiments, the input sample is a tissue. In some embodiments the input sample includes, but is not limited to epithelial tissue, connective tissue, muscular tissue, and/or nervous tissue. [0008] In some embodiments, the input sample is harvested from an animal. In some embodiments, the input sample is harvested from a human.

[0009] In some embodiments, the input sample is harvested from a plant. In some embodiments, the input sample includes, but is not limited to epidermal tissue, vascular tissue, ground tissue, meristematic tissue, simple permanent tissue, and/or complex permanent tissue. In some embodiments the input sample is harvested from mineral tissue.

[0010] In some embodiments, the pathogen is a bacterium, a virus, a protozoan, a parasite, a mold, or a fungus.

[0011] In some embodiments, the input samples are deposited into a receptacle for processing. In some embodiments, the receptacle is a microwell plate. In some embodiments, the receptacle is a microfluidic device. In some embodiments, the receptacle is a chip. In some embodiments, the receptacle is a tube or series of connected tubes, such as a PCR tube strip. In some embodiments, the receptacle is of a custom design to segregate different samples and optimize downstream performance. In some embodiments, the receptacle is disposable. In some embodiments, the receptacle is reusable.

[0012] In some embodiments, the depletion of host intracellular RNA/DNA begins with lysis of the host cells. In some embodiments, this method of lysis targets only host cells and not pathogens.

[0013] In some embodiments, host cells are lysed using methods including, but not limited to chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding. [0014] In some embodiments, the methods used to lyse the host cells are optimized to selectively target host cells and not pathogens. In some embodiments, this may include, but is not limited to altering reagent concentrations, levels of applied forces, temperatures, treatment duration, volumes, pressures, currents, and/or any other parameters within these protocols that may be adjusted.

[0015] In some embodiments, the lysis buffer used to lyse the host cells may comprise sodium dodecyl sulfate, Triton X-100, NP-40, Tween, cetyltrimethylammonium bromide, CHAPS, sodium deoxycholate, octylthioglucoside, octyl-beta-glucoside, and/or Brij-35.

[0016] In some embodiments, the host cells are lysed with a surfactant. In some embodiments, the surfactant is saponin. In some embodiments, the concentration of saponin used is optimized to target the selective lysis of host cells.

[0017] In some embodiments, depletion of host intracellular DNA/RNA follows the lysis step.

[0018] In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate by pipette. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the process of minicolumn purification. In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the process of phenol-chloroform extraction. In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the use of DNAses and RNAses. In other embodiments, host intracellular DNA/RNA is depleted through the use of other established methods for DNA/RNA depletion. [0019] In some embodiments, depletion of host extracellular DNA/RNA follows the depletion of the host intracellular DNA/RNA.

[0020] In some embodiments, host extracellular DNA/RNA is depleted with standard methods using propidium monoazide.

[0021] In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate by pipette. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the process of minicolumn purification. In some embodiments, host extracellular DNA/RNA is depleted through methods based on the process of phenol-chloroform extraction. In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the use of DNAses and RNAses. In other embodiments, host extracellular DNA/RNA is depleted through the use of other established methods for DNA/RNA depletion.

[0022] In some embodiments, any remaining host DNA/RNA is treated with magnetic beads to separate from the rest of the solution and washed off.

[0023] In some embodiments, pathogens are lysed following host DNA/RNA depletion.

[0024] In some embodiments, pathogens are lysed using the sporeLYSE method. In some embodiments, this may include, but is not limited to altering reagent concentrations, temperatures, treatment duration, volumes, pressures, currents, and/or any other parameters within these protocols that may be adjusted. In some embodiments, pathogens are lysed using the sporeLYSE method and one or more supplemental methods. In some embodiments, these supplemental may methods include, but are not limited to chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding.

[0025] In some embodiments, pathogens are lysed using one or more methods including, but not limited to chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding.

[0026] In some embodiments, pathogen DNAis extracted following lysis with standard methods using magnetic beads and a series of wash cycles.

[0027] In some embodiments, pathogen DNA is extracted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate by pipette. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, pathogen DNA is extracted through standard methods using the process of minicolumn purification. In some embodiments, pathogen DNAis extracted through standard methods using the process of phenol-chloroform extraction. In other embodiments, pathogen DNA is extracted through the use of other established methods for DNA extraction.

[0028] In some embodiments, the pathogen DNA library is prepared for sequencing. In some embodiments, this preparation step is contingent on the method of sequencing.

[0029] In some embodiments, the pathogen DNAis fragmented as part of the library preparation. In some embodiments, adapters and/or tags are added to the fragmented strands. In some embodiments, these adapters and/or tags are sample-specific to enable identification during multiplexed sequencing.

[0030] In some embodiments, the prepared pathogen DNA library is sequenced using nanopore sequencing. [0031] In some embodiments, the prepared pathogen DNA library is sequenced using a method including, but not limited to single-molecule real-time sequencing, ion semiconductor sequencing, Pyrosequencing, and/or chain termination sequencing.

[0032] In some embodiments, the sequence results are analyzed using established bioinformatics methods to identify pathogens present in the same. In some embodiments, the sequence results are analyzed using established bioinformatics methods to phenotypically and/or genetically characterize pathogens in the sample. In some embodiments, the sequence results are analyzed using established bioinformatics methods to characterize the antibiotic resistance of pathogens in the sample.

[0033] In some embodiments, the bioinformatic analysis is performed remotely through cloud computing. In some embodiments, the bioinformatic analysis is performed locally.

[0034] In some embodiments, the results of the bioinformatic analysis are processed and presented to the user in an easily accessible format.

[0035] In some embodiments, an automated device performs the steps following sample input, including, but not limited to the dispensing of reagents; pipetting of samples; disposal of waste; transfer of samples to different wells, lanes, reservoirs, channels, tubes, chips, plates, experimental devices; addition and/or removal of magnetic beads; application of magnetic fields; thermocycling; priming flow cells; incubating; and/or shaking.

[0036] In some embodiments, the user assists with some of the steps following sample input, including but not limited to adding reagents to reservoirs; adding pipette tips; emptying waste reservoirs; priming and/or flushing fluidic channels. [0037] In some embodiments, the steps are performed using different wells of a microarray plate, such that the sample is transferred to different wells depending on the experimental needs of the step. In some embodiments, these needs include, but are not limited to buffer conditions, reagent concentrations, temperature, magnetic field, pressure, and/or force.

[0038] The foregoing and other features of the disclosure will become more apparent from the following detailed description of several embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present disclosure will become better understood from the detailed description and the drawings, wherein:

[0040] FIG. l is a flow chart representing one embodiment of the methods and systems wherein a sample is added, intracellular and extracellular host DNA/RNA is depleted, pathogen DNA is extracted, prepared for sequencing, sequenced, and the results are analyzed and presented to the user.

[0041] FIG. 2 is a schematic representation of one embodiment of a microarray plate, wherein wells for performing each method step are identified.

[0042] FIG. 3 is a flow chart representing one embodiment of a workflow for a large liquid handling robot performing each method step.

[0043] FIG. 4 illustrates an exemplary liquid handling robot system.

[0044] FIG. 5 illustrates a diagram of an example system utilized in characterization of nonspecific pathogens in a sample.

[0045] FIG. 6 illustrates a diagram of an exemplary environment in which some embodiments may operate. DETAILED DESCRIPTION

[0046] In this specification, reference is made in detail to specific embodiments of the invention. Some of the embodiments or their aspects are illustrated in the drawings.

[0047] For clarity in explanation, the invention has been described with reference to specific embodiments, however it should be understood that the invention is not limited to the described embodiments. On the contrary, the invention covers alternatives, modifications, and equivalents as may be included within its scope as defined by any patent claims. The following embodiments of the invention are set forth without any loss of generality to, and without imposing limitations on, the claimed invention. In the following description, specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In addition, well known features may not have been described in detail to avoid unnecessarily obscuring the invention.

[0048] In addition, it should be understood that steps of the exemplary methods set forth in this exemplary patent can be performed in different orders than the order presented in this specification. Furthermore, some steps of the exemplary methods may be performed in parallel rather than being performed sequentially. Also, the steps of the exemplary methods may be performed in a network environment in which some steps are performed by different computers in the networked environment.

[0049] Some embodiments are implemented by a computer system. A computer system may include a processor, a memory, and a non-transitory computer-readable medium. The memory and non-transitory medium may store instructions for performing methods and steps described herein. [0050] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and it is not intended to mean that the compositions and methods exclude elements that are not recited. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of shall mean excluding more than a trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention. The singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of compounds, and a reference to "a molecule" is a reference to one or more molecules. Similarly, reference to “comprising a therapeutic agent” includes one or a plurality of such therapeutic agents. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. For example, the phrase “A or B” refers to A, B, or a combination of both A and B. Furthermore, the various elements, features and steps discussed herein, as well as other known equivalents for each such element, feature or step, can be mixed and matched by one of ordinary skill in this art to perform methods in accordance with principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in particular examples. All numerical designations, e.g., pH, temperature, time, concentration, amounts, and molecular weight, including ranges, are approximations which are varied (+) or (-) by 10%, 1%, or 0.1%, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0051] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All references cited herein are incorporated by reference in their entirety.

[0052] In some examples, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments are to be understood as being modified in some instances by the term "about" or "approximately." For example, "about" or "approximately" can indicate +/- 20% variation of the value it describes. Accordingly, in some embodiments, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties for a particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. [0053] To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0054] About: A term used to indicate a variation in value by +/-10% of the value, or optionally +1-5% of the value, or in some embodiments, by +/-1% of the value.

[0055] Agitate or Agitation: A mechanical movement that may include, but is not limited to, rotating, vibrating, vortexing, swirling, shaking, ultrasonicating, stirring, or any movement that causes mixing. Mechanical movements include movements performed by hand or by a rotator.

[0056] Amino Acids: organic compounds containing an amino functional group (-NH2) and a carboxyl functional group (-COOH), along with a side chain (R group) specific to each amino acid. About 500 naturally occurring amino acids are known. They can be classified according to the core structural functional groups' locations as alpha- (a-), beta- (b-), gamma- (g-) or delta- (d) amino acids, their polarity, or side chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur, etc.). In the form of proteins, amino acid residues form collagen, which is the main component of human muscles and connective tissues. Amino acids also participate in a number of processes, such as neurotransmitter transport and biosynthesis. Twenty of the proteinogenic amino acids are encoded directly by triplet codons in the genetic code and are known as "standard" amino acids. Many proteinogenic and non-proteinogenic amino acids have biological functions. Glutamate and gamma-amino-butyric acid (GABA) are neurotransmitters. Hydroxyproline, a major component of the connective tissue collagen, is synthesized from proline. Glycine is also a main component of collagen and a biosynthetic precursor to porphyrins. Carnitine is used in lipid transport. Nine proteinogenic amino acids are defined "essential amino acids" for humans because they cannot be produced from other compounds by the human body and must be taken in as food. [0057] Amino Acid Sequence: A sequence of amino acid residues corresponding to a protein or a polypeptide, or to a fragment or variant thereof having the protein or polypeptide function.

[0058] Analog: A compound having a structure similar to another, but differing from it, for example, in one or more atoms, functional groups, or substructure. Analog polypeptides include those substantially similar in structure and having the same biological activity to a naturally occurring molecule. Analogs may differ in the composition of their amino acid sequences compared to the naturally-occurring polypeptide from which the analog is derived, based on one or more mutations involving (i) deletion of one or more amino acid residues at one or more termini of the polypeptide and/or one or more internal regions of the naturally- occurring polypeptide; (ii) insertion or addition of one or more amino acids at one or more termini or internal regions of the polypeptide; or (iii) substitution of one or more amino acids for other amino acids in the naturally-occurring polypeptide sequence. Substitutions are conservative or non-conservative based on the physico-chemical or functional relatedness of the amino acid that is being replaced and the amino acid replacing it. Conservatively modified nucleic acids include nucleic acids, which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.

[0059] Antagonist: A molecule that, upon binding to a cell receptor, competes and/or interferes with one or more ligands binding the same receptor, and thus reduces or prevents a response elicited by those ligands.

[0060] Antibiotic: A chemical substance capable of treating bacterial infections by inhibiting the growth of, or by destroying existing colonies of bacteria and other microorganisms. [0061] Cell: A living biological cell, its progeny or potential progeny, which may be identical or non-identical to the parent cell.

[0062] Coding Region: A portion of the nucleic acid, which is transcribed and translated into a polypeptide or protein.

[0063] Conservative Substitution: A mutation in an amino acid sequence, wherein one or more amino acids is substituted by another amino acid having highly similar properties. Examples include, but are not limited to, one or more amino acids comprising nonpolar or aliphatic side chains, such as glycine, alanine, valine, leucine, isoleucine or proline, which can be substituted for one another; one or more amino acids comprising polar, uncharged side chains, such as serine, threonine, cysteine, methionine, asparagine or glutamine, which can be substituted for each other; one or more amino acids comprising aromatic side chains, such as phenylalanine, tyrosine or tryptophan, which can be substituted for each other; one or more amino acids comprising positively charged side chains, such as lysine, arginine or histidine, which can be substituted for each other; one or more amino acids comprising negatively charged side chains, such as aspartic acid or glutamic acid, which can be substituted for each other. For example, conservative substitutions for leucine include alanine, isoleucine, valine, phenylalanine, tryptophan, methionine, and cysteine, and conservative substitutions for asparagine include arginine, lysine, aspartate, glutamate, and glutamine.

[0064] Contacting: Placement in direct physical association; includes both in solid and liquid form.

[0065] Culture Medium: A nutrient suspension containing salts, amino acids, growth regulators, sugars, and buffers used to grow cells in vitro. [0066] Effective amount: The amount of an active agent (alone or with one or more other active agents) sufficient to induce a desired response, such as to prevent, treat, reduce and/or ameliorate a condition. Effective amounts of an active agent, alone or with one or more other active agents, can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the condition in the subject or measuring the level of one or more molecules associated with the condition to be treated.

[0067] Fragment or Variant: Any functional fragment, variant, derivative or analog of a polynucleotide, polypeptide or biomolecule that possesses an in vivo or in vitro activity that is characteristic of the polynucleotide, polypeptide or biomolecule. The biomolecule can be an antibody that is characterized by antigen-binding activity, or an enzyme characterized by the ability to catalyze a biochemical reaction.

[0068] Gene: A coding region operably linked to one or more sequences that regulate the expression of a polypeptide or protein. A gene includes un-translated regulatory regions of DNA, such as promoters, enhancers, and repressors, preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences, such as introns and exons, between individual coding regions. Regulatory sequences may be linked to the gene before the coding sequence (5' non-coding sequences) or after the coding sequence (3' non-coding sequences). “Native” or “wild-type” genes are genes found in nature. A modified or mutant gene is a gene that comprises a modified sequence relative to the corresponding native gene. A “chimeric gene” is a gene that comprises regulatory and coding sequences that are not found together in nature. A chimeric gene may comprise regulatory sequences and coding sequences that are derived from different (heterologous) sources, or regulatory sequences and coding sequences derived from the same source (homologous), but arranged in a manner different than is found in nature.

[0069] Hybridization: A process used to identify a particular sequence of interest, and by which a nucleic acid strand joins with a complementary strand through hydrogen bonding at complementary bases. Hybridization assays are run under stringent conditions. Stringent conditions are defined by concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. Stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.

[0070] Hydrocolloid: A substance that produces a gel when dispersed in water.

[0071] Hydrogel: A water-swellable polymeric matrix that can absorb a substantial amount of water to form elastic gels. The matrix is a three-dimensional network of macromolecules held together by covalent or non-covalent crosslinks. Upon placement in an aqueous environment, dry hydrogels swell to the extent allowed by the degree of cross-linking.

[0072] Hydrogel Composition: A composition that either contains a hydrogel or is entirely composed of a hydrogel. Thus, "hydrogel compositions" encompass not only hydrogels per se but also compositions that comprise a hydrogel and one or more non-hydrogel components or compositions, e.g., hydrocolloids, which contain a hydrophilic component (which may contain or be a hydrogel) distributed in a hydrophobic phase.

[0073] Hydrophilic: A polymer, substance or compound that is capable of absorbing more than 10%/w of water at 100% relative humidity (RH). [0074] Hydrophobic: A polymer, substance or compound that is capable of absorbing no more than 1%/w of water at 100% relative humidity (RH).

[0075] Hygroscopic: A polymer, substance or compound that is capable of absorbing more than 20 w% of water at 100% relative humidity (RH).

[0076] Linker: A sequence connecting two polynucleotide sequences or two polypeptide sequences. A peptide linker may consist of from about 1 to about 100, or from about 1 to about 50 amino acids in length. Multiple peptide linkers may be used. The linker may be attached to the target nucleic acid molecule or target peptide via covalent bonding, non-covalent bonding, ionic bonding, hydrophobic interactions or any combination thereof. The type and length of the linker can be selected to optimize tethering, proximity, flexibility, rigidity, or orientation. The attachment can be reversible or non-reversible. Linkers may be linear, branched, bifunctional, trifunctional, homobifunctional linkers, or heterobifunctional linkers.

[0077] Lipophilic: A substance or compound that has an affinity for a non-polar environment compared to a polar or aqueous environment.

[0078] Liposome: A spherical vesicle containing one or more lipid bilayers composed of phospholipids, such as phosphatidylcholine and phoshatidylethanolamine, which surround a hydrophilic core. Liposomes can be used as vehicles for transport of nutrients and drugs, and can be prepared by sonication of biological membranes.

[0079] Microbial Cell: A cell of a microorganism within the fungal, yeast or bacterial families, which grows over a wide range of temperature, pH values, and solvent tolerances. Examples of suitable microbial host strains include, but are not limited to, fungal or yeast species, such as Aspergillus, Penicillium, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Kluyveromyces; bacterial species, such as Agrobacterium, Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia coli, Salmonella, Pseudomonas, Bacteroides, and Cornyebacterium; and algae species, such as Chlamydomonas.

[0080] Nucleic Acid: A naturally occurring or synthetic DNA, RNA, DNA-RNA hybrid, single- stranded or double-stranded oligonucleotide or polynucleotide, and which hybridizes to a complementary nucleic acid sequence. Nucleic acids include, but are not limited to, deoxyribonucleotides, ribonucleotides and polymers thereof in either single- or double- stranded form, nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides, conservatively modified variants thereof, such as variants containing degenerate codon substitutions, and complementary sequences. Sequences with degenerate codon substitutions include sequences in which the third position of one or more codons is substituted with mixed-base and/or deoxyinosine residues.

[0081] Oil: Any fatty substance that is in viscous liquid form at room temperature (25° C) and at atmospheric pressure (760 mmHg). Oils are hydrophobic and lipophilic, have a high carbon and hydrogen content and are usually flammable and surface active. Oils may be animal, vegetable, or petrochemical in origin, and may be volatile or non-volatile. Oils may be used for food, fuel, medical purposes, and for the manufacture of paints and plastics.

[0082] Operably Linked: An association between nucleic acid sequences on a single nucleic acid molecule such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Operational linkage can also be engineered through synthetic means, including those disclosed herein.

[0083] pH Adjuster or Modifier: A molecule or buffer used to achieve desired pH control in a formulation. Exemplary pH modifiers include acids (e.g., acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, phosphoric acid, sorbic acid, succinic acid, tartaric acid, basic pH modifiers (e.g., magnesium oxide, tribasic potassium phosphate), and pharmaceutically acceptable salts thereof.

[0084] Phospholipid: a class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic. The structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are joined together by a glycerol molecule.

[0085] Polypeptide: A naturally occurring or synthetic peptide, oligopeptide, polypeptide, gene product, expression product, or protein comprising an amino acid sequence, wherein the amino acids are joined to each other by peptide bonds or modified peptide bonds.

[0086] Purification or Purify: Any technique or method that increases the degree of purity of a substance of interest, such as an enzyme, a protein, or a compound, from a sample comprising the substance of interest. Methods for purification are well known in the art. Non-limiting examples of purification methods include silica gel column chromatography, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography including, but not limited to, cation and anion exchange chromatography, free-flow- electrophoresis, high performance liquid chromatography (HPLC), and differential precipitation. In the context of this application, for example, a Sepharose resin column may be used to purify the substance of interest, such as an enzyme or a product of an enzymatic reaction.

[0087] Recovery: A process involving isolation and collection of a product from a reaction mixture. The product can be a compound, such as a protein, a nucleotide, or a lipid. Methods to recover products are well known in the art. Recovery methods may include, but are not limited to, chromatography, such as silica gel chromatography and HPLC, activated charcoal treatment, filtration, distillation, precipitation, drying, chemical derivation, and any combinations thereof.

[0088] Sequence Identity or Homology: Refers to two or more nucleic acid or amino acid sequences or subsequences that are identical or share a specified percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence. The percent identity between two given sequences can be calculated using an algorithm such as BLAST. The phrase "substantially identical," in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90%, about 95%, about 98%, about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Preferably, substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.

[0089] Transcription Factor: A protein involved in the process of transcribing DNA into RNA. Transcription factors bind to specific sequences of DNA to modify transcription and, consequently, synthesis of messenger RNA (mRNA). Modification is direct when the transcription factor binds through its DNA binding domain to a promoter region of DNA and to RNA Polymerase II to start transcription, or indirect if the transcription factor binds one or more cofactors through its activator domain. Post-translational modifications of transcription factors - including, but limited to interactions with small molecule inducers - may affect the DNA-binding capacity of a transcription factor or alter its association with transcriptional coactivator and repressor complexes, resulting in changes in gene expression profile. Transcription factors may physically interact with each other to form homodimers or heterodimers, resulting in inhibition or enhancement of transcriptional activity. These interactions allow crosstalk between different signal transduction pathways at the level of gene expression. Simultaneous activation of several transcription factors is generally necessary for maximal gene expression.

[0090] Transformation: The process by which a nucleic acid sequence of interest, or a fragment or variant of the nucleic acid sequence of interest that encodes a polypeptide having the same functional activity of a polypeptide encoded by the nucleic acid sequence of interest is transferred into a host organism or the genome of a host organism, resulting in genetically stable inheritance. In some cases, the transformation may be intentionally transient. Transformed host organisms are also known as "recombinant", "transgenic" or "transformed" organisms. Thus, isolated polynucleotides disclosed herein can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. A construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region, such as a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression, a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a poly-adenylation signal.

[0091] Under conditions sufficient to: A phrase that is used to describe any environment that permits the desired reaction to take place.

[0092] Vector: A nucleic acid, plasmid or virus used to transfer coding information to a host cell. A "cloning vector" is a small piece of DNA into which a DNA fragment is inserted to be transcribed. Typically the vector contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. The DNA sequence in the expression vector is operably linked to appropriate expression control sequences, including a promoter, to direct RNA synthesis and protein expression. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome-binding site for translation initiation and a transcription terminator, and include appropriate sequences for amplifying expression. In addition, the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Useful selectable markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. The vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, Ti-mediated gene transfer, calcium phosphate transfection, DEAE-Dextran-mediated transfection, lipofection, or electroporation. Examples of vectors include, but are not limited to, viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA, such as vaccinia and adenovirus, PI -based artificial chromosomes, yeast plasmids, Bacillus vectors, and Aspergillus vectors. Examples of bacterial vectors include, but are not limited to, pQE vectors, pBluescript plasmids, pNH vectors, and lambda-ZAP vectors. Examples of eukaryotic vectors include pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 vectors.

[0093] SYSTEMS FOR CHARACTERIZATION OF NONSPECIFIC PATHOGENS IN A SAMPLE

[0094] Disclosed herein are systems for the automated detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample. These novel systems work by coupling a “one size fits all” approach to the extraction of pathogen DNA from a sample to the high speed and accuracy of nanopore sequencing. Because these systems sidestep the limitations of traditional culturing approaches, it’s both significantly faster and able to identify a greater breadth of pathogens, as it’s not restricted by the culturability of the sample. Moreover, by sequencing the pathogens, users will have immediate access to phenotypic data, including antibiotic resistance, which would traditionally take additional lab work to accomplish.

[0095] At their core, the disclosed systems comprise two primary elements: a system to process the input samples, and software to process the sequence data. The sample processing system comprises an automated liquid handling robot that is designed to carry out all necessary steps between user input and the sequencing of pathogen DNA. The software will then process the sequence data to determine the taxonomic identity of the pathogens, as well as phenotypic characteristics, and then present this analysis to the user in a readily accessible format.

[0096] In some embodiments, the input comprises a receptacle and one or more samples to be tested. In some embodiments, the receptacle is a microwell plate. In some embodiments, the receptacle is a microfluidic device. In some embodiments, the receptacle is a chip. In some embodiments, the receptacle is a tube or series of connected tubes, such as a PCR tube strip. In some embodiments, the receptacle is of a custom design to segregate different samples and optimize downstream performance. In some embodiments, the receptacle is disposable. In some embodiments, the receptacle is reusable.

[0097] In some embodiments, different wells, chambers, channels and/or tubes of the receptacle are configured to perform different reaction steps. In some embodiments, the receptacle may come preloaded with experimental elements including, but not limited to reagents, magnetic beads, filters, columns, enzymes, tags, and/or buffers. In some embodiments, these elements are added by the automated liquid handling robot after sample input.

[0098] In some embodiments, some wells, chambers, channels, and/or tubes will are configured for specific steps based on the geometry of the receptacle. In some embodiments, some wells, chambers, channels and/or tubes may be more readily subjected to magnetic fields, thermocycling, shaking, pressures, currents, and/or other parameters.

[0099] In some embodiments, an automated liquid handling robot may transfer the input sample to different wells, chambers, channels, and/or tubes as the sample is processed. In some embodiments, an automated liquid handling robot may dispense or remove reagents, buffers, and/or enzymes; remove and/or dispose waste; mix compositions in a well, chamber, channel, and/or tube; and add and/or remove magnetic beads. [0100] In some embodiments, the system may further comprise a thermocycling element to provide precision temperature control within specific wells, chambers, channels, and/or tubes of the receptacle.

[0101] In some embodiments, the system may further comprise a magnetic element to control the magnetic field applied to specific wells, chambers, channels, and/or tubes of the receptacle.

[0102] In some embodiments, the system may further comprise an element to shake and/or agitate the receptacle.

[0103] In some embodiments, the system may further comprise a microcentrifuge.

[0104] In some embodiments, the system may further comprise disposable and/or reusable pipette tips.

[0105] In some embodiments, the input sample is a biofluid. In some embodiments the input sample includes, but is not limited to blood, saliva, excreta, lymph, perilymph, endolymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, serous fluid, joint fluid, interstitial fluid, and/or transcellular fluid.

[0106] In some embodiments, the input sample is a tissue. In some embodiments the input sample includes, but is not limited to epithelial tissue, connective tissue, muscular tissue, and/or nervous tissue.

[0107] In some embodiments, the input sample is harvested from an animal. In some embodiments, the input sample is harvested from a human.

[0108] In some embodiments, the input sample is harvested from a plant. In some embodiments, the input sample includes, but is not limited to epidermal tissue, vascular tissue, ground tissue, meristematic tissue, simple permanent tissue, and/or complex permanent tissue. In some embodiments the input sample is harvested from mineral tissue.

[0109] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle may be used for lysis of host cells.

[0110] In some embodiments, lysis buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for lysis of host cells. In some embodiments, lysis buffers may be added by the liquid handling robot.

[0111] In some embodiments, saponin lyses the host cells. In some embodiments, the concentration of saponin is optimized to target the selective lysis of host cells.

[0112] In some embodiments, the system may further comprise elements for lysing host cells. In some embodiments, these may include, but are not limited to elements for chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding.

[0113] In some embodiments, these lysis buffers may comprise sodium dodecyl sulfate, Triton X- 100, NP-40, Tween, cetyltrimethylammonium bromide, CHAPS, sodium deoxycholate, octylthioglucoside, octyl-beta-glucoside, and/or Brij-35.

[0114] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle are used for depletion of intracellular host DNA/RNA. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for depletion of intracellular host DNA/RNA are the same wells, chambers, channels, and/or tubes of the receptacle used for host cell lysis. [0115] In some embodiments, depletion enzymes, reagents, and/or buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for depletion of intracellular host DNA/RNA. In some embodiments, depletion enzymes, reagents, and/or buffers may be added by the liquid handling robot.

[0116] In some embodiments, DNAses and/or RNAses may deplete intracellular host DNA/RNA.

[0117] In some embodiments, cold ethanol or isopropanol may deplete intracellular host DNA/RNA.

[0118] In some embodiments, the liquid handling robot may remove the precipitate.

[0119] In some embodiments, the system may further comprise elements for depletion of intracellular host DNA/RNA. In some embodiments, these may include, but are not limited to elements for minicolumn purification and/or phenol-chloroform extraction.

[0120] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle are used for depletion of extracellular host DNA/RNA. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for depletion of extracellular host DNA/RNA are the same wells, chambers, channels, and/or tubes of the receptacle used for intracellular host DNA/RNA depletion.

[0121] In some embodiments, depletion enzymes, reagents, and/or buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for depletion of extracellular host DNA/RNA. In some embodiments, depletion enzymes, reagents, and/or buffers may be added by the liquid handling robot.

[0122] In some embodiments, propidium monoazide may deplete host extracellular DNA/RNA. [0123] In some embodiments, magnetic beads may separate any remaining host DNA/RNA from the rest of the solution.

[0124] In some embodiments, the system may further comprise elements for depletion of intracellular host DNA/RNA. In some embodiments, these may include, but are not limited to elements for minicolumn purification, DNAses/RNAses, and/or phenol-chloroform extraction.

[0125] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle are be used for lysis of pathogens. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for lysis of pathogens are the same wells, chambers, channels, and/or tubes of the receptacle used for extracellular host DNA/RNA depletion.

[0126] In some embodiments, lysis enzymes, reagents, and/or buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for pathogen lysis. In some embodiments, lysis enzymes, reagents, and/or buffers may be added by the liquid handling robot.

[0127] In some embodiments, sporeLYSE will be added to lyse pathogens. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for pathogen lysis are subjected to thermacycle control.

[0128] In some embodiments, the system may further comprise elements for lysis of pathogens. In some embodiments, these may include, but are not limited to elements for chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding. [0129] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle are used for purification of pathogen DNA. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for purification of pathogen DNA are the same as the wells, chambers, channels, and/or tubes of the receptacle used for lysis of pathogens.

[0130] In some embodiments, purification enzymes, magnetic beads, reagents, and/or buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for pathogen lysis. In some embodiments, purification enzymes, reagents, and/or buffers may be added by the liquid handling robot.

[0131] In some embodiments, magnetic beads separate pathogen DNA from the rest of the solution. In some embodiments, buffers separate the DNA from the magnetic beads.

[0132] In some embodiments, one or more wells, chambers, channels, and/or tubes of the receptacle are used for library preparation of pathogen DNA. In some embodiments, the wells, chambers, channels, and/or tubes of the receptacle used for library preparation of pathogen DNA are the same as the wells, chambers, channels, and/or tubes of the receptacle used for purification of pathogen DNA.

[0133] In some embodiments, library preparation enzymes, tags, reagents, and/or buffers may be present in the wells, chambers, channels, and/or tubes of the receptacle used for pathogen lysis. In some embodiments, library preparation enzymes, reagents, and/or buffers may be added by the liquid handling robot.

[0134] In some embodiments, enzymes fragment the pathogen DNA.

[0135] In some embodiments, adapters and/or tags are added to the fragmented pathogen DNA. [0136] In some embodiments the system further comprises elements for sequencing DNA. In some embodiments, the system may comprise one or more nanopore sequencers. In some embodiments, the system may comprise elements for single-molecule real-time sequencing, ion semiconductor sequencing, Pyrosequencing, and/or chain termination sequencing.

[0137] In some embodiments, the system comprises a network connection.

[0138] In some embodiments, the system comprises a cloud computing platform capable of analyzing sequence data using established bioinformatic methods.

[0139] In some embodiments, the system comprises a local computing platform capable of analyzing sequence data using established bioinformatic methods.

[0140] In some embodiments, the system comprises an interface for displaying the results of the bioinformatic analysis to the user.

[0141] Referring to FIG. 1, an example method is described and performed according to the disclosure herein. Abiofluid sample is provided (step 101). Host cell lysis is performed (step 103), for example, with the application of Saponin 102. Depletion of host DNA/RNA is performed (step 105), for example, with the application of propidium monoazide (104). Pathogen lysis is performed (step 107), for example, with the application of SporeLYSE. Extraction of pathogen DNAis performed (step 109), for example, using magnetic beads (108). Fragmentation of pathogen is performed (step 111), for example, with the application of fragmentation enzymes (110). Adapters (112) are added to pathogen DNA (step 113). Sequencing of DNA is performed (step 115), for example, by a Nonapore sequencer (114). Analysis of the sequences is performed (step 117), for example, by analysis software (116). Then the software (116) produces results indicative of one or more identified pathogens. [0142] Referring to FIG. 2, a schematic representation of one embodiment of a microarray plate, wherein wells for performing each method step are identified. The microarray plate, for example, may have multiple sample wells (i.e., Sample 1 through Sample 12). At the top row, magnetic beads may be utilized (205). At the next row, DNA library may be utilized. At the next row, DNA tags (207) may be utilized. At the next row, fragmentation enzymes may be utilized (206). At the next row, SporeLYSE (204) may be utilized. At the next row, propidium monoazide (203), may be utilized. At the next row, saponin (202) may be utilized. At the bottom, row a sample (201) may be provided. The liquid handling robotic may be programmed to performed the operations described herein.

[0143] Referring to FIG. 3, an example method is described and performed according to the disclosure herein. Abiofluid sample is provided (step 101). Saponin (102) is transferred and the sample to well. The saponin (102) and sample are mixed together and allowed to incubate for a period of time (step 103). Propidium monoazide (104) is transferred and sample to well, and are mixed together and allowed to incubate for a period of time (step 105). The mixed sample may then be transferred to a centrifuge tube, and then spun down to remove DNA (step 106). SporeLYSE may then be applied to the sample, and mixed together and allowed to incubate for a period of time (step 108). Magnetic beads (109) may be transferred to the sample, and the beads washed, DNA may be removed (step 110). Fragmentation enzymes (111), and adapters/tags (112) may be applied, and mixed together for a period of time and allowed to incubate (step 113). The prepared DNA is transferred to a nanapore sequence (step 114) for sequencing..

[0144] Methods for Regulatory Element Characterization [0145] Also provided herein are methods for the detection, sequencing, identification, and/or characterization of nonspecific pathogens in a sample. These novel methods work by coupling a “one size fits all” approach to the extraction of pathogen DNA from a sample to the high speed and accuracy of nanopore sequencing. Because these methods sidestep the limitations of traditional culturing approaches, it’s both significantly faster and able to identify a greater breadth of pathogens, as it’s not restricted by the culturability of the sample. Moreover, by sequencing the pathogens, users will have immediate access to phenotypic data, including antibiotic resistance, which would traditionally take additional lab work to accomplish.

[0146] In some embodiments, the methods comprise: (a) user input of sample; (b) depletion of the host intracellular RNA/DNA; (c) depletion of the host extracellular RNA/DNA; (d) extraction of the pathogen DNA; (e) preparation of the DNA library for sequencing; (f) sequencing; (g) sequence analysis; and (h) delivery of results to the user.

[0147] In some embodiments, the input sample is a biofluid. In some embodiments the input sample includes, but is not limited to blood, saliva, excreta, lymph, perilymph, endolymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, serous fluid, joint fluid, interstitial fluid, and/or transcellular fluid.

[0148] In some embodiments, the input sample is a tissue. In some embodiments the input sample includes, but is not limited to epithelial tissue, connective tissue, muscular tissue, and/or nervous tissue.

[0149] In some embodiments, the input sample is harvested from an animal. In some embodiments, the input sample is harvested from a human. [0150] In some embodiments, the input sample is harvested from a plant. In some embodiments, the input sample includes, but is not limited to epidermal tissue, vascular tissue, ground tissue, meristematic tissue, simple permanent tissue, and/or complex permanent tissue. In some embodiments the input sample is harvested from mineral tissue.

[0151] In some embodiments, the pathogen is a bacterium, a virus, a protozoan, a parasite, a mold, or a fungus.

[0152] In some embodiments, the host cells of the input sample are lysed prior to intracellular DNA/RNA depletion.

[0153] In some embodiments, the host cells of the input sample are lysed with saponin. In some embodiments, the concentration of saponin used is optimized to target the selective lysis of host cells and not the pathogens.

[0154] In some embodiments, the concentration of saponin used is 0.025%. In some embodiments, the concentration of saponin used is 0.015-0.035%. In some embodiments, the concentration of saponin used is 0.001-0.015%. In some embodiments, the concentration of saponin used is 0.035-0.5%. In some embodiments, the concentration of saponin used is greater than 0.5%.

[0155] In some embodiments, the saponin is diluted in deionized water.

[0156] In some embodiments, host cells are lysed using methods including, but not limited to chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding.

[0157] In some embodiments, the methods used to lyse the host cells are optimized to selectively target host cells and not pathogens. In some embodiments, this may include, but is not limited to altering reagent concentrations, levels of applied forces, temperatures, treatment duration, volumes, pressures, currents, and/or any other parameters within these protocols that may be adjusted.

[0158] In some embodiments, the lysis buffer used to lyse the host cells may comprise Sodium dodecyl sulfate, Triton X-100, NP-40, Tween, Cetyltrimethylammonium bromide, CHAPS, Sodium deoxycholate, Octylthioglucoside, Octyl-beta-glucoside, and/or Brij-35.

[0159] In some embodiments, the sample is incubated for a period of time following addition of lysis agent, buffer, enzyme, or surfactant. In some embodiments, this period of time is 0-10 minutes. In some embodiments, this period of time is greater than 10 minutes.

[0160] In some embodiments, the incubation is at room temperature. In some embodiments, the incubation is at 37°C.

[0161] In some embodiments, the sample is shaken, agitated, vortexed, and/or stirred following addition of lysis agent, buffer, enzyme, or surfactant.

[0162] In some embodiments, depletion of host intracellular DNA/RNA follows the lysis step. In some embodiments, depletion of host intracellular DNA/RNA is concurrent with the lysis of host cells.

[0163] In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the use of DNAses and RNAses. In some embodiments, this includes the addition of DNAses and/or RNAses, the addition of DNAse buffer and/or RNAse buffer, mixing, and incubation. In some embodiments, the incubation period is 0-20 minutes. In some embodiments, the incubation period is 20-40 minutes. In some embodiments, the incubation period is longer than 40 minutes. In some embodiments, the incubation is at room temperature. In some embodiments, the incubation is at 37°C. [0164] In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the process of minicolumn purification. In some embodiments, host intracellular DNA/RNA is depleted through standard methods using the process of phenol-chloroform extraction. In other embodiments, host intracellular DNA/RNA is depleted through the use of other established methods for DNA/RNA depletion.

[0165] In some embodiments, depletion of host extracellular DNA/RNA follows the depletion of the host intracellular DNA/RNA. In some embodiments, depletion of host extracellular DNA/RNA and intracellular DNA/RNA are concurrent steps. In some embodiments, depletion of host extracellular DNA/RNA is not a mandatory step.

[0166] In some embodiments, host extracellular DNA/RNA is depleted with standard methods using propidium monoazide. In some embodiments, propidium monoazide is added to the sample at a final concentration of 0-25mM. In some embodiments, propidium monoazide is added to the sample at a final concentration of 25-50mM. In some embodiments, propidium monoazide is added to the sample at a final concentration of greater than 50mM.

[0167] In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the process of minicolumn purification. In some embodiments, host extracellular DNA/RNA is depleted through methods based on the process of phenol-chloroform extraction. In some embodiments, host extracellular DNA/RNA is depleted through standard methods using the use of DNAses and RNAses. In other embodiments, host extracellular DNA/RNA is depleted through the use of other established methods for DNA/RNA depletion.

[0168] In some embodiments, there is an incubation period following the addition of host extracellular DNA/RNA depletion reagents, enzymes, buffers, and/or chemicals. In some embodiments, the incubation period is 0-20 minutes. In some embodiments, the incubation period is 20-40 minutes. In some embodiments, the incubation period is longer than 40 minutes. In some embodiments, the incubation is at room temperature. In some embodiments, the incubation is at 37°C.

[0169] In some embodiments, the last depletion step is the removal of host DNA/RNA from the sample. In some embodiments, host DNA/RNA is removed from the sample using standard methods of magnetic bead DNA/RNA extraction. In some embodiments, host DNA/RNA is removed from the sample using standard methods of centrifugation and separation of the supernatant from the pellet.

[0170] In some embodiments, pathogen lysis follows the depletion and removal of host DNA/RNA.

[0171] In some embodiments, the sporeLYSE method is used for pathogen lysis. In some embodiments, this includes an incubation period of 0-5 minutes. In some embodiments, this includes an incubation period of 5-20 minutes. In some embodiments, the incubation period is 20-40 minutes. In some embodiments, the incubation period is longer than 40 minutes. In some embodiments, the incubation is at 70-80°C. In some embodiments, the incubation is at temperatures greater than 80°C. In some embodiments, the incubation is at temperatures less than 70°C. In some embodiments, a buffer is added following this incubation period, which is followed by a second incubation period.

[0172] In some embodiments, one or more supplemental methods may also be used for pathogen lysis. In some embodiments, supplemental may methods include, but are not limited to chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, and/or manual grinding.

[0173] In some embodiments, pathogen DNAis extracted following pathogen lysis.

[0174] In some embodiments, pathogen DNA is extracted with standard methods using standard methods of magnetic bead DNA extraction. In some embodiments, the magnetic beads are added to the sample and followed by a series of wash steps, wherein the DNA is extracted by changing the buffer conditions to release it from the magnetic beads.

[0175] In some embodiments, pathogen DNA is extracted through standard methods using the addition of cold ethanol or isopropanol and removal of the precipitate by pipette. In some embodiments, this may include additional reagents and buffers and centrifugation. In some embodiments, pathogen DNA is extracted through standard methods using the process of minicolumn purification. In some embodiments, pathogen DNAis extracted through standard methods using the process of phenol-chloroform extraction. In other embodiments, pathogen DNA is extracted through the use of other established methods for DNA extraction.

[0176] In some embodiments, the pathogen DNA library is prepared for sequencing following extraction. In some embodiments, this preparation step is contingent on the method of sequencing. In some embodiments, the pathogen DNA is washed prior to library preparation. [0177] In some embodiments, the pathogen DNAis fragmented as part of the library preparation. In some embodiments, pathogen DNA is fragmented using enzymes. In some embodiments, pathogen DNA is fragmented using centrifugation and filters and/or orifices that shear the DNA. In some embodiments, pathogen DNA is fragmented using sonication. In some embodiments, the ideal fragment length is 3-8kb.

[0178] In some embodiments, adapters and/or tags are added to the fragmented strands. In some embodiments, the adapters and/or tags are added using PCR. In some embodiments, adapters and/or tags are added using ligation. In some embodiments, adapters and/or tags are added using transposase enzymes. In some embodiments, these adapters and/or tags are sample- specific to enable identification during multiplexed sequencing.

[0179] In some embodiments, the prepared pathogen DNA library is sequenced using nanopore sequencing.

[0180] In some embodiments, the prepared pathogen DNA library is sequenced using a method including, but not limited to single-molecule real-time sequencing, ion semiconductor sequencing, Pyrosequencing, and/or chain termination sequencing.

[0181] In some embodiments, the sequence results are analyzed using established bioinformatics methods to identify pathogens present in the same. In some embodiments, the sequence results are analyzed using established bioinformatics methods to phenotypically and/or genetically characterize pathogens in the sample. In some embodiments, the sequence results are analyzed using established bioinformatics methods to characterize the antibiotic resistance of pathogens in the sample. [0182] In some embodiments, the bioinformatic analysis is performed remotely through cloud computing. In some embodiments, the bioinformatic analysis is performed locally.

[0183] In some embodiments, the results of the bioinformatic analysis are processed and presented to the user in an easily accessible format.

[0184] Advantages of the Disclosed Processes

[0185] The present invention overcomes limitations inherent in other methods for detection, identification, and/or characterization of pathogens. Traditional approaches have relied on culturing of samples, which is slow, limited to facilities with culturing equipment, restricted to the subset of pathogens that can be cultured, and requires further testing to determine antibiotic resistance, among other phenotypic traits. The present invention, on the other hand, offers a fast, automated, point-of-care operable system and method that can provide full phenotypic analysis and isn’t restricted to pathogens that can be cultured.

[0186] Practical Applications

[0187] The present invention can be used for multiple applications, including but not limited to, point of care diagnostic tests for infectious disease, point of care diagnostic tests for rare genetic diseases, point of care pharmacogenetic tests, point of care companion diagnostic tests for gene therapies, and/or microbiome tests.

[0188] EXAMPLES

[0189] Example 1: Identification of infectious agent and its antibiotic resistance

[0190] Multiple biofluid samples are taken from a patient, including blood, urine, and saliva. The samples are diluted to an appropriate concentration and added to individual wells in a microwell plate. The plate is placed inside an OT-2 liquid handling robot. [0191] As part of the first DNA depletion step, the liquid handling robot takes an aliquot of each biofluid sample and mixes it with a buffer in new individual wells. The liquid handling robot pipettes saponin into each well to reach a final concentration of 0.025%. Samples are agitated for 10 seconds and incubated at room temperature for 5 minutes. The liquid handling robot adds lOx Turbo DNAse buffer to a final concentration of lx, followed by the addition of 2pl of Turbo DNAse. Samples are gently mixed and incubated at 37°C for 30 minutes. Samples are spun down and are resuspended in sterile distilled water.

[0192] In a second DNA depletion step, the liquid handling robot adds propidium monoazide to each sample well to a final concentration of IOmM. The samples are mixed then incubated in the dark at room temperature for 5 minutes. Following this, samples are transferred to unique wells on the microplate where they are chilled to 4°C and illuminated with fluorescent light for 25 minutes, with some intermittent agitation. Samples are then spun down, washed off, and resuspended in 200m1 of sterile distilled water.

[0193] To lyse the pathogens, the samples are transferred to new wells on the microplate. The liquid handling robot adds 200m1 sporeLYSE Lysis Buffer and 40m1 sporeLYSE Lysis Reagent. The samples are heated to 70°C for 20 minutes, followed by the addition of 40m1 sporeLYSE N-Lysis Buffer and incubation at 4°C for 10 minutes.

[0194] To extract pathogen DNA, the samples are transferred to new wells containing magnetic beads, which bind to DNA present in the sample. After mixing, a magnetic field is applied to the wells, immobilizing the DNA-bound beads against one side of the well. The wells are then washed to remove the rest of the sample, followed by the addition of elution buffer to release the purified pathogen DNA. [0195] To prepare the DNA library, the liquid handling robot transfers the DNA to new wells, along with adapters, transposase buffers, and transposase enzymes, which fragments the DNA and adds adapters to the ends of the fragments.

[0196] To prepare for sequencing, the flow cells for Oxford Nanopore Sequencers inside the OT- 2 are primed. The liquid handling robot loads the prepared pathogen DNA libraries onto the flow cells and the sequencing protocol is run.

[0197] Data generated from the Oxford Nanopore Sequencers is uploaded to a cloud-based bioinformatics pipeline, which uses BLAST to determine taxonomy and antibiotic resistance data about the sequences. Results are curated and returned to the end user in a readily accessible format.

[0198] Example 2: Microbiome genetic analysis

[0199] A stool sample is taken from a patient. The sample is homogenized and aliquoted into a well into a tube.

[0200] As part of the first DNA depletion step, the sample is mixed with a buffer, followed by the addition of saponin into the tube to reach a final concentration of 0.025% The sample is vortexed for 10 seconds and incubated at room temperature for 5 minutes. Following this, lOx Turbo DNAse buffer is added to a final concentration of lx, followed by the addition of 2pl of Turbo DNAse. The sample is gently mixed by pipetting and incubated at 37°C for 30 minutes. The sample is spun down and is resuspended in sterile distilled water.

[0201] In a second DNA depletion step, propidium monoazide is added to the sample to a final concentration of IOmM. The sample is mixed then incubated in the dark at room temperature for 5 minutes. Following this, the sample is transferred to a new tube and put on ice and illuminated with fluorescent light for 25 minutes, with some intermittent shaking. The sample is spun down, washed off, and resuspended in 200pl of sterile distilled water.

[0202] To lyse the microbiota, the sample is transferred to a new tube. 200m1 sporeLYSE Lysis Buffer and 40m1 sporeLYSE Lysis Reagent are added. The sample is heated to 70°C for 20 minutes, followed by the addition of 40m1 sporeLYSE N-Lysis Buffer and incubation at 4°C for 10 minutes.

[0203] To extract microbiota DNA, magnetic beads are added to the sample, which bind to the DNA. After mixing, a magnetic field is applied to the tube, immobilizing the DNA-bound beads against one side of the tube. The tube is then washed to remove the rest of the sample, followed by the addition of elution buffer to release the purified microbiota DNA.

[0204] To prepare the DNA library, the DNA is fragmented using sonication. The fragmented DNA is then added to a tube along with adapters, ligation buffers, and ligases, which add adapters to the ends of the fragments.

[0205] To prepare for sequencing, a flow cell for an Oxford Nanopore Sequencer is primed. The prepared microbiota DNA library is loaded onto the flow cell and the sequencing protocol is run.

[0206] Data generated from the Oxford Nanopore Sequencer is uploaded to a cloud-based bioinformatics pipeline, which uses BLAST to determine taxonomy and a mutation profile of the microbiome. Results are curated and returned to the end user in a readily accessible format.

[0207] Example 3: Test for rare genetic diseases [0208] A blood sample is taken from a patient. The sample is diluted to an appropriate concentration and added to a well in a microwell plate. The plate is placed inside a liquid handling robot.

[0209] As part of the first DNA extraction step, the liquid handling robot mixes the sample with a SDS lysis buffer and adds Proteinase K. The sample is stirred for 10 seconds and incubated at 56°C for 1 hour.

[0210] In a second DNA extraction step, the liquid handling robot adds an equal volume of phenol: chloroform: isoamyl alcohol solution (25:24:1), gently mixing the solution for 3 minutes, then centrifuging the sample at 10,000g at 4°C for 10 minute. The upper aqueous layer is removed and added to a new well.

[0211] To prepare the DNA library, the liquid handling robot adds adapters, transposase buffers, and transposase enzymes, which fragments the DNA and adds adapters to the ends of the fragments.

[0212] To prepare for sequencing, a flow cell for an Oxford Nanopore Sequencer inside the liquid handling robot is primed. The liquid handling robot loads the prepared DNA library onto the flow cell and the sequencing protocol is run.

[0213] Data generated from the Oxford Nanopore Sequencer is uploaded to a cloud-based bioinformatics pipeline, which uses sequence alignment algorithms to determine point mutations within the sequences, which are cross-referenced to databases of genetic diseases. Results are curated and returned to the end user in a readily accessible format.

[0214] Example Liquid Handling Robotic System [0215] FIG. 4 illustrates an exemplary liquid handling robot system. An example liquid handling robot that may be used to perform operations as described herein can be the Opentrons OT-2 lab robot. Information about the Opentrons OT-2 liquid handling robot system may be found at www.opentrons.com

[0216] Example System

[0217] FIG. 5 illustrates a diagram of an example system 500 utilized for characterization of nonspecific pathogens in a sample. The system 500 may include a Robotic Control Module 504, a Data Acquisition Module 506, a Machine Learning Module 510, a Data Processing Module 512 and a User Interface Module 516.

[0218] While the databases 520, 522, 524 are displayed separately, the databases and information maintained in a database 520, 522, 524 may be combined together or further separated in a manner that promotes retrieval and storage efficiency and/or data security. The databases may include information related to protocol and procedures to control the liquid handling robot, storing obtained sample results and the determined pathogens found in the processed samples.

[0219] The Robotic Control Module 504 may perform functionality related to a controlling the liquid handling robot. For example, the module 504 may control the operations of the handling robot.

[0220] The Data Acquisition Module 506 may perform functionality related to handling communication and receipt and transfer of data received by the system via user interfaces, and for obtaining data from and/or sending data to a server based system for additional processing of sample data. [0221] The Machine Learning Module 510 may perform functionality related to evaluating sample processing result for determining the occurrence of a particular pathogen.

[0222] The Data Processing Module 512 may perform functionality related to additional processing of sample data.

[0223] The User Interface Module 516 may perform functionality related to rendering and display of information as described herein.

[0224] The User Device 540 may have an Application Engine 542 and a User Interface 544. It is understood that the system 500 may further include one or more additional modules for performing, or supporting performance of, any operation(s), step(s), act(s), instruction(s) and process(es) described herein.

[0225] FIG. 6 illustrates an example machine of a computer system within which a set of instructions, for causing the machine to perform any one or more of the methodologies discussed herein, may be executed. In alternative implementations, the machine may be connected (e.g., networked) to other machines in a LAN, an intranet, an extranet, and/or the Internet. The machine may operate in the capacity of a server or a client machine in client- server network environment, as a peer machine in a peer-to-peer (or distributed) network environment, or as a server or a client machine in a cloud computing infrastructure or environment.

[0226] The machine may be a personal computer (PC), a tablet PC, a set-top box (STB), a Personal Digital Assistant (PDA), a cellular telephone, a web appliance, a server, a network router, a switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine. Further, while a single machine is illustrated, the term “machine” shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein.

[0227] The example computer system 600 includes a processing device 602, a main memory 604 (e.g., read-only memory (ROM), flash memory, dynamic random access memory (DRAM) such as synchronous DRAM (SDRAM) or Rambus DRAM (RDRAM), etc.), a static memory 606 (e.g., flash memory, static random access memory (SRAM), etc.), and a data storage device 618, which communicate with each other via a bus 630.

[0228] Processing device 602 represents one or more general-purpose processing devices such as a microprocessor, a central processing unit, or the like. More particularly, the processing device may be a complex instruction set computing (CISC) microprocessor, reduced instruction set computing (RISC) microprocessor, very long instruction word (VLIW) microprocessor, or processor implementing other instruction sets, or processors implementing a combination of instruction sets. Processing device 602 may also be one or more special-purpose processing devices such as an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a digital signal processor (DSP), network processor, or the like. The processing device 602 is configured to execute instructions 626 for performing the operations and steps discussed herein.

[0229] The computer system 600 may further include a network interface device 608 to communicate over the network 620. The computer system 600 also may include a video display unit 610 (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)), an alphanumeric input device 612 (e.g., a keyboard), a cursor control device 614 (e.g., a mouse), a graphics processing unit 622, a signal generation device 616 (e.g., a speaker), graphics processing unit 622, video processing unit 628, and audio processing unit 632.

[0230] The data storage device 618 may include a machine-readable storage medium 624 (also known as a computer-readable medium) on which is stored one or more sets of instructions or software 626 embodying any one or more of the methodologies or functions described herein. The instructions 626 may also reside, completely or at least partially, within the main memory 604 and/or within the processing device 602 during execution thereof by the computer system 600, the main memory 604 and the processing device 602 also constituting machine-readable storage media.

[0231] Some portions of the preceding detailed descriptions have been presented in terms of algorithms and symbolic representations of operations on data bits within a computer memory. These algorithmic descriptions and representations are the ways used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art. An algorithm is here, and generally, conceived to be a self-consistent sequence of operations leading to a desired result. The operations are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic signals capable of being stored, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like.

[0232] It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. Unless specifically stated otherwise as apparent from the above discussion, it is appreciated that throughout the description, discussions utilizing terms such as “identifying” or “determining” or “executing” or “performing” or “collecting” or “creating” or “sending” or the like, refer to the action and processes of a computer system, or similar electronic computing device, that manipulates and transforms data represented as physical (electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage devices.

[0233] The present disclosure also relates to an apparatus for performing the operations herein. This apparatus may be specially constructed for the intended purposes, or it may comprise a general purpose computer selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a computer readable storage medium, such as, but not limited to, any type of disk including floppy disks, optical disks, CD- ROMs, and magnetic-optical disks, read-only memories (ROMs), random access memories (RAMs), EPROMs, EEPROMs, magnetic or optical cards, or any type of media suitable for storing electronic instructions, each coupled to a computer system bus.

[0234] Various general purpose systems may be used with programs in accordance with the teachings herein, or it may prove convenient to construct a more specialized apparatus to perform the method. The structure for a variety of these systems will appear as set forth in the description above. In addition, the present disclosure is not described with reference to any particular programming language. It will be appreciated that a variety of programming languages may be used to implement the teachings of the disclosure as described herein.

[0235] The present disclosure may be provided as a computer program product, or software, that may include a machine-readable medium having stored thereon instructions, which may be used to program a computer system (or other electronic devices) to perform a process according to the present disclosure. A machine-readable medium includes any mechanism for storing information in a form readable by a machine (e.g., a computer). For example, a machine- readable (e.g., computer-readable) medium includes a machine (e.g., a computer) readable storage medium such as a read only memory (“ROM”), random access memory (“RAM”), magnetic disk storage media, optical storage media, flash memory devices, etc.

[0236] In the foregoing disclosure, implementations of the disclosure have been described with reference to specific example implementations thereof. It will be evident that various modifications may be made thereto without departing from the broader spirit and scope of implementations of the disclosure as set forth in the following claims. The disclosure and drawings are, accordingly, to be regarded in an illustrative sense rather than a restrictive sense.

[0237] In the foregoing disclosure, implementations of the disclosure have been described with reference to specific example implementations thereof. It will be evident that various modifications may be made thereto without departing from the broader spirit and scope of implementations of the disclosure as set forth in the following claims. The disclosure and drawings are, accordingly, to be regarded in an illustrative sense rather than a restrictive sense.

Claims

CLAIMS What is claimed is:

1. A method for detecting, sequencing, identifying, and characterizing nonspecific pathogens in one or more samples, wherein the method comprises: a) selectively lysing host cells using saponin; b) depleting host RNA/DNA using propidium monoazide; c) lysing pathogens using sporeLYSE; d) extraction of the pathogen DNA; e) preparing the pathogen DNA library for sequencing through fragmentation and addition of adapters; f) sequencing the pathogen DNA library using nanopore sequencing; and g) analysing the sequence data.

2. The method of Claim 1, wherein the one or more samples comprises a biofluid.

3. The method of Claim 2, wherein the biofluid comprises blood, saliva, excreta, lymph, perilymph, endolymph, cerebrospinal fluid, peritoneal fluid, pleural fluid, amniotic fluid, serous fluid, joint fluid, interstitial fluid, transcellular fluid, or any combination thereof.

4. The method of Claim 1, wherein the one or more samples comprises a tissue.

5. The method of Claim 1, wherein the tissue comprises epithelial tissue, connective tissue, muscular tissue, nervous tissue, or any combination thereof.

6. The method of Claim 1, wherein the sample is harvested from an animal.

7. The method of Claim 6, wherein the animal is a human.

8. The method of Claim 1, wherein the selective lysis of host cells further comprises the use of chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, manual grinding, or any combination thereof.

9. The method of Claim 1, wherein the depletion of host RNA/DNA further comprises the use of DNAses, RNAses, magnetic bead DNA extraction, phenol-chloroform extraction, ethanol or isopropanol extraction, minicolumn purification, or any combination thereof.

10. The method of Claim 1, wherein the pathogen lysis further comprises the use of chemical lysis, acoustic lysis, mechanical lysis, liquid homogenization, thermal lysis, enzymatic lysis, electrical lysis, laser lysis, osmotic shock, manual grinding, or any combination thereof.

11. The method of Claim 1, wherein the extraction of pathogen DNA comprises magnetic bead DNA extraction, phenol-chloroform extraction, ethanol or isopropanol extraction, minicolumn purification, or any combination thereof.

12. The method of Claim 1, wherein the DNA fragmentation comprises the use of enzymes, DNA shearing columns, soni cation, or any combination thereof.

13. The method of Claim 1, wherein the adding of adapters to the DNA comprises the use of PCR, ligation enzymes, transposase enzymes, or any combination thereof.

14. The method of Claim 1, wherein the analysis the sequence data is performed remotely through a cloud computing platform.

15. The method of Claim 1, wherein the selective lysis of host cells, depletion of host RNA/DNA, lysis of pathogens, extraction of the pathogen DNA, preparation of the pathogen DNA library for sequencing, and/or all intermediate steps are performed by a liquid handling robot.

16. A system for automatically detecting, sequencing, identifying, and characterizing nonspecific pathogens in one or more samples, comprising: a) one or more receptacles; b) a liquid handling robot; c) reagents for host cell lysis comprising saponin; d) reagents for depletion of host DNA/RNA comprising propidium monoazide; e) reagents for pathogen lysis comprising sporeLYSE; f) one or more nanopore sequencers; and g) sequence analysis software.

17. The system of Claim 16, wherein the one or more receptacles comprise a microwell plate, a microfluidic device, a chip, a tube or any combination thereof.

18. The system of Claim 16, wherein the reagents for host cell lysis further comprise sodium dodecyl sulfate, Triton X-100, NP-40, Tween, cetyltrimethylammonium bromide, CHAPS, sodium deoxycholate, octylthioglucoside, octyl-beta-glucoside, Brij-35, or any combination thereof.

19. The system of Claim 16, wherein the reagents for host DNA/RNA depletion further comprise DNAses, RNAses, phenol-chloroform, ethanol, isopropanol, or any combination thereof.

20. The system of Claim 16, wherein the system further comprises reagents for the preparation of a DNA library for sequencing.

21. The system of Claim 20, wherein the reagents comprise restriction enzymes, ligation enzymes, transposase enzymes, adapters, or any combination thereof.

22. The system of Claim 16, wherein the system further comprises magnetic beads.

23. The system of Claim 16, wherein the system further comprises one or more magnetic actuators.

24. The system of Claim 16, wherein the system further comprises one or more thermocyclers.

25. The system of Claim 16, wherein the system further comprises one or more centrifuges.

26. The system of Claim 16, wherein the system further comprises one or more sonicators.

27. The system of Claim 16, wherein the system further comprises one or more DNA shearing columns.