JP2024063020A - A versatile method for extracting nucleic acid molecules from a diverse population of one or more types of microorganisms in a sample. - Google Patents

Abstract

A method is provided for extracting genetic material from a diverse population of one or more types of microorganisms in a sample. The microorganisms may be prokaryotic or eukaryotic, and may include bacteria, archaea, fungi, protozoa, helminths, parasites, viruses, phages, and others. Extraction may be from a single sample, and subsequent identification may be via molecular methods such as qPCR, PCR, RFLP, SSCP, allele-specific PCR, targeted sequencing, pull-down sequencing, full shotgun sequencing, or other methods. Also provided is a method that includes extracting nucleic acid molecules from various organisms, such as fungi (i.e., Saccharomyces spp.), animal cells (bovine), plants (e.g., barley), and from the gut of a human subject, performing metagenomics analysis therefrom, and determining a probiotic treatment or dietary guideline for the subject based on the metagenomics analysis.Selected Figure: FIG.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119 to U.S. Patent Application No. 62/651,620, filed April 2, 2018. The disclosure of the prior application is considered a part of this application and is incorporated by reference into the disclosure of this application.

FIELD OF THEINVENTION The present invention relates generally to genomic analysis, and more specifically to methods for extracting and analyzing food-associated nucleic acid molecules from diverse populations of microorganisms in biological samples.

Background Information Approximately 100 trillion microorganisms live on and in the human body, far outnumbering the approximately 10 trillion human cells in the human body. These normally harmless viruses, bacteria, and fungi are called symbiotic or mutualistic organisms. Symbiotic or mutualistic organisms help keep our bodies healthy in a variety of ways. Collectively, all the symbiotic and pathogenic microorganisms living on and in the body are called the microbiome, and their equilibrium and associated metabolome are closely related to the health of an individual and vice versa.

Advances in nucleic acid sequencing have created the opportunity to rapidly and accurately identify and profile the microbiome inhabiting the gut and subcutaneous tissues. An optimal flora also interacts with the host immune system in a synergistic manner, further multiplying its health benefits. The associated metabolome of an individual can also be profiled by mass spectrometry-based systems or using genomics-based metabolomic modeling and flux balance analysis, which can be used to create a healthy metabolomic profile. All these methodologies can be used to analyze the complexity of microbial populations.

The present invention is directed to methods of extracting nucleic acid molecules from diverse microbial populations in biological, environmental, nutraceutical, or other ecological microbial heterogeneous population samples, and methods of using the nucleic acids or extracts through processing steps and analysis to determine the customization of probiotics to individuals. Processing steps specific to the present invention include RNA or DNA cleanup, fragmentation, separation, or digestion; library or nucleic acid preparation for downstream applications such as PCR, qPCR, digital PCR, or sequencing; pre-processing for bioinformatics QC, filtering, alignment, or data separation; metagenomics or human genome bioinformatics pipelines for microbial species taxonomic assignment; and other organism alignment, identification, and variant interpretation.

The present invention also describes a versatile method for using the sample for DNA extraction and determination of food intake based on food DNA sequences from a database of meat, plants, fruits, vegetables, and/or microorganisms contained with these organisms. Disclosed herein is a method for extracting genetic material from a diverse population of one or more types of cells or cellular components in a sample and determining the breakdown of food and nutrients ingested for improving health and preventing disease.

Thus, in one aspect, the invention provides a method of preparing a sample for analysis. The method includes: a) mixing the sample with a first lysis solution comprising a detergent, e.g., SDS, and a chelating agent, e.g., EDTA; b) adding a second lysis solution having lysozyme to the mixture of step a); and c) adding a third lysis solution comprising a chaotropic agent, e.g., urea, lithium acetate, guanidine hydrochloride, etc., to the mixture of step b). The pretreatment step may include physical lysis, which may be used to further optimize nucleic acid yield. Examples of mechanical lysis include sonication, bead mixing, and bead mill homogenization.

In a similar aspect, the method includes a) mixing a sample, such as a stool sample, with a liquid nitrogen solution; b) adding a first lysis solution, the first lysis solution including a detergent and a chelating agent, such as SDS, and a chelating agent, such as EDTA; and c) adding a second lysis solution, the second lysis solution including a chaotropic agent, such as urea, lithium acetate, guanidine hydrochloride. Pretreatment steps can include physical lysis, which can be used to further optimize nucleic acid yields. Examples of mechanical lysis include sonication, bead mixing, and bead mill homogenization.

In another aspect, the present invention provides a method of determining food intake of a subject. The method includes: a) extracting genetic material from a stool sample obtained from the subject, the genetic material being extracted according to the method of the present disclosure; and b) subjecting the genetic material extracted from the first sample to metagenomic analysis to determine the food intake of the subject. In an embodiment, the method further includes treating the subject with a probiotic or food based on the analysis of the food intake.

In another aspect, the present invention provides a method of monitoring probiotic treatment of a subject. The method includes: a) extracting genetic material from any microorganisms present in a first sample obtained from the subject, the genetic material being extracted according to the method of the present disclosure; b) subjecting the genetic material extracted from the first sample to metagenomic analysis; c) treating the subject with a probiotic and then extracting genetic material from any microorganisms present in a second sample obtained from the subject similar to the extraction of the genetic material from the first sample; d) performing metagenomic analysis on the genetic material extracted from the second sample; and e) comparing the results of the metagenomic analysis of the first sample to the metagenomic analysis of the second sample.

In yet another aspect, the present invention provides a method comprising calculating a probiotic score from the probiotic organisms detected in the gut, with or without additional chemical or genetic testing.

In yet another aspect, the invention provides a method that includes calculating a microbiome score, which is used to assess whether the microbiome is in a dysbiosis, neutral, or stable state.

The present invention further provides a computing system including a memory and one or more processors coupled to the memory, the one or more processors configured to perform operations to implement the method of the present invention.

The present invention also provides an automated platform for carrying out the methods of the present invention.

The present invention provides an all-in-one method for extracting nucleic acids from a diverse flora of microorganisms from biological, environmental, dietary supplement, or other ecological microbial heterogeneous population samples.

In embodiments, the present invention can be used to determine the composition and relative abundance of microorganisms in probiotic and environmental samples through analysis of their respective nucleic acids. DNA is purified and used downstream for nucleic acid analysis (particularly metagenomics analysis where the genomes of two or more species/subspecies are identified).

[The present invention 1001]
a) mixing the sample with a liquid nitrogen solution;
b) adding a first solution, the first solution including a surfactant and a chelating agent;
and c) adding a second lysis solution, said second lysis solution comprising a chaotropic agent.
[The present invention 1002]
The method of claim 10, wherein said mixing of said sample with a liquid nitrogen solution further comprises grinding the mixture of said sample and said liquid nitrogen.
[The present invention 1003]
1001. The method of claim 1001, wherein said first lysis solution further comprises one or more buffers, one or more mild detergents, and/or one or more proteases.
[The present invention 1004]
1001. The method of claim 1001, wherein the surfactant in the first lysis solution comprises SDS.
[The present invention 1005]
The method of claim 1004, wherein the concentration of SDS is about 10%.
[The present invention 1006]
1001. The method of claim 1001, wherein the chaotropic agent comprises lithium acetate.
[The present invention 1007]
The method of claim 1006, wherein the mixture of the sample, liquid nitrogen, said first lysis solution, and said second lysis solution is further subjected to a heat shock treatment.
[The present invention 1008]
The method of claim 1001, wherein the sample is subjected to a pretreatment step prior to treatment with the first lysis solution, the pretreatment step inducing germination of any bacterial and/or fungal spores present in the sample.
[The present invention 1009]
The method of any one of claims 10 to 15, wherein said pretreatment step comprises mixing said sample with Tween-80.
[The present invention 1010]
The method of claim 1001, further comprising a mechanical treatment step to cause physical lysis, said mechanical treatment step comprising sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, or a combination thereof.
[The present invention 1011]
The method of claim 1001, further comprising subjecting any extracted genetic material to metagenomics analysis.
[The present invention 1012]
The method of claim 10, wherein said sample is obtained from the intestine of a subject.
[The present invention 1013]
The method of claim 1011, wherein the metagenomics analysis identifies one or more foods ingested by the subject.
[The present invention 1014]
The method of any one of claims 10 to 11, wherein the metagenomics analysis identifies one or more macronutrients ingested by the subject.
[The present invention 1015]
The method of any one of claims 1 to 10, further comprising determining a probiotic treatment for said subject based on said metagenomics analysis.
[The present invention 1016]
The method of any one of claims 1 to 10, further comprising determining a dietary guideline for the subject based on the metagenomics analysis.
[The present invention 1017]
1. A method for determining food intake in a subject, comprising:
a) extracting genetic material from a stool sample obtained from said subject, said genetic material being extracted according to the present invention;
b) subjecting the genetic material extracted from the first sample to metagenomics analysis to determine the food intake of the subject.
[The present invention 1018]
The method of claim 1017, further comprising treating said subject with a probiotic or food based on said food intake analysis.
[The present invention 1019]
The method of the present invention 1017, wherein the metagenomic analysis involves the use of a database having genomic data of organisms, the database being useful for identifying such organisms.
[The present invention 1020]
The method of the present invention 1019, wherein the database can be treated as whole genomes, as k-mers of various lengths that are common to higher orders and specific to particular ones, or as other means of barcoding genomes and matching them to sequencing results.
[The present invention 1021]
Metagenomic analysis
The method of the present invention 1017 includes pre-processing of sequencing information selected from removing duplications, removing adapter sequencing, removing 5' or 3' sequencing to improve base call quality and include only base calls of a certain quality (i.e., Q20 or higher), filtering human reads, creating or separating paired reads, and limiting read overlap.
[The present invention 1022]
Aligning the sequencing information to the database using software or a system, where the sequencing information may be split into k-mers of a particular length, or may be used as complete fragments, or may be scaffolded and aligned to a large reference genome; or
The method of the present invention 1019, wherein the metagenomic analysis includes other methods for generating reports of identified organisms, relative abundance of identified organisms, genome size, total aligned fragments, unique fragments aligned to strain, species, genus, family, order, class, phylum, kingdom, or domain.
[The present invention 1023]
The method of the present invention 1017, wherein the microbiome profile allows for the identification of a disease, disorder, or specific signature indicative of dysbiosis, and probiotic and/or nutritional supplementation treatments can be applied to modulate the microbiome to improve the underlying profile.
[The present invention 1024]
The method of the present invention 1023, wherein groups are created based on demographic, phenotypic, or diagnostic information and a barcode is assigned to the profile group.
[The present invention 1025]
The method of the present invention 1024, wherein statistical analysis is used to determine how closely an individual's microbiome profile is related to known groups in the database.
[The present invention 1026]
1. A method for monitoring probiotic treatment in a subject, comprising:
a) extracting genetic material from any microorganisms present in a first sample obtained from said subject, said genetic material being extracted according to the invention 1001;
b) subjecting the genetic material extracted from the first sample to metagenomics analysis;
c) treating the subject with a probiotic and then extracting genetic material from any microorganisms present in a second sample obtained from the subject similar to the extraction of the genetic material from the first sample;
d) performing metagenomics analysis on the genetic material extracted from the second sample; and
e) comparing the results of the metagenomics analysis of the first sample with the results of the metagenomics analysis of the second sample.
[The present invention 1027]
The method of claim 1026, further comprising modifying said probiotic treatment to obtain a desired microbial population in said subject.
[The present invention 1028]
The method of claim 1026 further comprising analysis of metabolomic markers to determine an appropriate probiotic treatment.
[The present invention 1029]
The method of claim 1026, wherein the probiotic treatment is administered after antibiotics, chemotherapy, use of pharmaceutical drugs, environmental changes, travel, ingestion of contaminants, intestinal or intestinal obstruction, stress, or other action that may result in the disruption of the microbial population.
[The present invention 1030]
The method of claim 1027, wherein modulating the microbiome reverts an individual to a previous population of microorganisms when said individual was known to be healthy.
[The present invention 1031]
The method of the present invention 1027, wherein the modulation of the microbiome by probiotics and/or prebiotics is to restore the microbiome profile to a normal gut as defined internally by a database or externally by a public database.
[The present invention 1032]
The method of the present invention 1031, wherein normal is defined as similar to the microbiome profile of a fecal material repository sample used for fecal transplantation but used as a probiotic/prebiotic to reverse dysbiosis.
[The present invention 1033]
A computing system comprising a memory and one or more processors coupled to the memory, the one or more processors configured to execute operations to perform the method of the present invention 1017.
[The present invention 1034]
A computing system comprising a memory and one or more processors coupled to the memory, the one or more processors configured to execute operations to perform the method of the present invention 1026.
[The present invention 1035]
An automated platform for carrying out the method of the present invention 1001.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. Any accompanying drawings are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

FIG. 1 is a schematic illustrating the presence of prevalence organisms in the human (high protein diet, >50 years old, supplement user) microbiome signature. FIG. 1 is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (viruses and phages) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (Archaea) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (fungi and other eukaryotes) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, vegetarian diet). FIG. 1 is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, non-vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (viruses and phages) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, non-vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (Archaea) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, non-vegetarian diet). FIG. 1 is a schematic illustrating the presence of high prevalence organisms (fungi and other eukaryotes) in the microbiome signature of humans (high carbohydrate diet, 18-50 years old, non-vegetarian diet). FIG. 1 is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) in the human (high milk protein diet, 0-2 years old, vegetarian non-breast-fed) microbiome signature. FIG. 1 is a schematic illustrating the presence of high prevalence organisms (viruses and phages) in the human (high milk protein diet, 0-2 years old, vegetarian non-breast-fed) microbiome signature. FIG. 1 is a schematic illustrating the presence of high prevalence organisms (Archaea) in the human (high milk protein diet, 0-2 years old, vegetarian non-breast-fed) microbiome signature. FIG. 1 is a schematic illustrating the presence of high prevalence organisms (fungi and other eukaryotes) in the human (high milk protein diet, 0-2 years old, vegetarian non-breast-fed) microbiome signature. FIG. 1 is a schematic showing the presence of lower prevalence organisms in human microbiome signatures and identification of opportunistic pathogens. FIG. 1 is a schematic showing typical probiotics detected in human microbiome signatures. FIG. 1 is a schematic showing typical probiotics detected in human microbiome signatures. FIG. 1 is a schematic graph showing individual relative abundances compared to the database mean for the normal population. 1 is a table showing organisms identified via the methods of the present invention from dietary supplement mixed cultures. 1 is a table listing the unique species classifications of various microorganisms stored in the database of the present invention. 1 shows exemplary demographic information from an individual in one embodiment of the present invention. 1 illustrates exemplary organisms detected in association with seafood in one embodiment of the present invention. 1 shows exemplary organisms detected in association with mammalian meat in one embodiment of the present invention. 1 illustrates exemplary organisms detected in association with grain in one embodiment of the present invention.

DETAILED DESCRIPTION OF THE PRESENTINVENTION The present invention provides a versatile method for extracting nucleic acid molecules from a diverse population of one or more types of microorganisms in a sample. The types of microorganisms include gram-positive bacteria, gram-positive bacterial spores, gram-negative bacteria, archaea, protozoa, parasitic helminths, algae, fungi, fungal spores, viruses, viroids, bacteriophages, and rotifers. In some embodiments, the diverse population is a plurality of different microorganisms of the same type, e.g., gram-positive bacteria. In some embodiments, the diverse population is a plurality of different types of microorganisms, e.g., bacteria (gram-positive bacteria, gram-positive bacterial spores, and/or gram-negative), fungi, viruses, and bacteriophages.

Because different types of microorganisms have different compositions and mechanisms for protecting their own genetic material, it is often difficult to extract genetic material from one type of microorganism without compromising the ability to also extract genetic material from another type of microorganism in the same biological sample. However, the present invention makes it possible to extract genetic material from different types of microorganisms in a sample without sacrificing the amount of genetic material that can be obtained from one type of microorganism by extracting genetic material from another type of microorganism in the same sample. According to the present invention, the sample containing the microorganism may be a biological sample, an environmental sample, an artificially created sample (e.g., a laboratory test or control sample, a sample of a probiotic composition or a food supplement, etc.). Examples of biological samples include tissue samples, blood samples, plasma samples, cerebrospinal fluid samples, urine samples, fecal samples, samples of material obtained from the digestive tract, biological secretions (e.g., semen, vaginal secretions, breast milk, tears, saliva, etc.), etc. A solid sample may be liquefied or mixed with a solution, and then the genetic material of the microorganisms present in the liquefied sample, mixture, or solution obtained from the mixture may be extracted according to the present invention. The extracted genetic material can be subjected to further processing and analysis, such as purification, amplification, and sequencing.

In some embodiments, the extracted genetic material is subjected to metagenomics analysis, for example to identify one or more types of microorganisms in the sample from which the genetic material was extracted. In additional embodiments, whole genome shotgun sequencing can be performed on extracted nucleic acid material prepared from human fecal samples. Preparation includes nucleic acid clean-up reactions to remove organic solvents, impurities, salts, phenols, and other process-inhibiting contaminants. Additional preparation includes nucleic acid library preparation from each sample, where gDNA undergoes modification and/or amplification to prepare the sample for sequencing on a sequencing platform such as massively parallel sequencing by synthesis, nanopore, long read, and/or CMOS electronic sequencing.

As disclosed herein, the method of the present invention allows the successful extraction of genetic material from one or more different types of microorganisms present in the same sample by subjecting the microorganisms to three different compositions in a specific order. The method according to the present invention comprises, first, lysing any Gram-negative bacteria present in the sample, followed by digesting the polysaccharide components of any yeast and bacterial cell walls present in the sample, and then disrupting with a chaotropic agent any cell walls that are intact after the second step.

Briefly, in one embodiment, the first step involves mixing the sample with a first lysis solution that includes a detergent (e.g., sodium dodecyl sulfate (SDS)) and a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)) to lyse any gram-negative bacteria present in the sample. The first lysis solution may further include one or more buffers (e.g., Tris), one or more mild detergents (e.g., Triton™ X-100), and/or one or more proteases (e.g., proteinase K).

After the first step, the sample is mixed with a second lysis solution containing lysozyme to digest the polysaccharide components of the yeast and bacterial cell walls present in the mixture. It is important that contact between the sample and the second lysis solution occurs after treatment of the sample with the first lysis solution, since lysozyme can inhibit the activity of the first lysis solution.

After treatment with the second lysis solution, a third lysis solution containing a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.) is added to the mixture to disrupt any cell walls not digested by the second lysis solution. The third lysis solution may contain a detergent such as SDS.

In some embodiments, both the first lysis solution and the third lysis solution contain SDS at a working concentration of 1-10% w/v. In some embodiments, after treatment with the third lysis solution, the mixture is further treated with a fourth lysis solution containing a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.) and proteinase K. In some embodiments where the chaotropic agent of the third lysis solution is lithium acetate, the mixture may then be subjected to a heat shock treatment and then treated with the fourth lysis solution.

In certain aspects, the disclosure below describes a versatile method for using stool samples for DNA extraction and determination of food intake based on food DNA sequences from a database of meat, plants, fruits, vegetables, and/or microorganisms contained with these organisms. Disclosed herein is a method for extracting genetic material from a diverse population of one or more types of cells or cellular components in a sample to determine the breakdown of food and nutrients ingested for improved health and prevention of disease.

In some embodiments, biological secretions (e.g., semen, vaginal secretions, breast milk, tears, saliva, blood, urine, etc.) are obtained from the digestive tract, etc. The solid sample may be liquefied or mixed with a solution, and then the genetic material of any food containing genetic material, such as plant (seedlings, leaves, cotyledons, seeds, endosperm, tissue culture callus, roots, etc.), animal, fungal, or protozoan foods, in the liquefied sample, mixture, or solution obtained from the mixture, can be extracted according to the present invention or other standard nucleic acid extraction protocols known in the art. In some embodiments, the extracted genetic material may be subjected to further processing and analysis, such as purification, amplification, and sequencing. In some embodiments, the extracted genetic material is subjected to metagenomics analysis, for example, to identify one or more types of organisms in the sample from which the genetic material was extracted.

In some embodiments, the databases utilized by metagenomic analysis are customized for the specific purpose of identifying and taxonomically assigning nucleic acids with relative abundances of organisms or components of organisms ingested by humans or other animals within appropriate lineages. In some embodiments, additional data tables or databases may be used as a lookup of the relative abundances of organisms to determine the macronutrient content of gut samples of organisms as representative of their diet. In some embodiments, this macronutrient breakdown may include fat, carbohydrate, protein, vitamins minerals, and any macronutrient subcomponents.

As disclosed herein, the methods of the invention allow for the successful extraction of genetic material from one or more different types of organisms, one or more cells of an organism, or a cell matrix or organelle present in the same sample by subjecting the sample to isolation, purification, or other methods for capturing nucleic acids. The methods according to the invention include, but are not limited to, lysing or disrupting any food cells in the sample, including any cell walls and cell membranes, digesting the polysaccharide or lignin components of any cell walls or membranes of any fungal, plant, mammalian, or protozoan cells present in the sample, and disrupting with a chaotropic agent any cell walls that are intact after the digestion step.

The invention includes physically disrupting the cell walls or membranes of food cells by liquid nitrogen flash freezing and immediate mechanical disruption or grinding to disrupt the cell walls and keep harmful cellular enzymes inactive prior to chemical lysis. The invention includes mixing the sample with a first lysis solution that includes a detergent (e.g., sodium dodecyl sulfate (SDS)) and a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)) to lyse any animal cells present in the sample. The first lysis solution may further include one or more buffers (e.g., Tris), one or more mild detergents (e.g., Triton™ X-100, cetyltrimethylammonium bromide), and/or one or more proteases (e.g., proteinase K). In certain embodiments, the first lysis solution includes SDS at a working concentration of 1-10% w/v. The invention includes mixing the sample with a second lysis solution that includes a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.). The second lysis solution may include a detergent, such as SDS. In certain exemplary embodiments, the first and second lysis solutions may be added in any particular order.

In some embodiments, the invention may include a step involving mixing the sample with a third lysis solution containing lysozyme to digest any fungal or bacterial cell wall polysaccharide components present in the mixture. In some embodiments, the mixture may be further treated with a fourth lysis solution containing a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, etc.) and proteinase K. In some embodiments where the chaotropic agent of the fourth lysis solution is lithium acetate, the mixture may then be subjected to a heat shock treatment and then treated with the fourth lysis solution. In certain exemplary embodiments, the third and/or fourth solutions may be added to the mixture at any time to destroy any cell walls not digested by any preceding lysis solution.

In some embodiments, if the sample has or is suspected of having bacterial and/or fungal spores, the sample may be subjected to a pretreatment step to induce germination of the cell walls of the spores prior to contact with the first lysis solution. The pretreatment step may include mixing the sample with a chemical such as a mild detergent, e.g., Tween-80, to induce germination, or incubating the sample under conditions (e.g., temperature) that induce germination. In some embodiments where germination is induced with a chemical, the chemical preferably does not inhibit, reduce, or alter the activity or effectiveness of the first, second, and third lysis solutions.

In some embodiments, the methods according to the invention may further include one or more mechanical processing steps that induce physical lysis by mechanical methods including sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, etc. In some embodiments, the mechanical processing step is performed prior to subjecting the sample to the first lysis solution.

In an embodiment, the method according to the invention is directed to the production and/or storage of bacteria, such as yeast (i.e., Saccharomyces spp.), Gram-negative bacteria (e.g., Acinetobacter spp.), Gram-positive bacteria (e.g., Bifidobacterium spp.), viruses (e.g., Sclerotinia spp.), spores (Bacillus spp.), helminths (e.g., tapeworms, Echinococcus spp.), and/or fungi (e.g., cephalosporins, cephalosporins, and/or fungi). Nucleic acid molecules can be extracted from a variety of microorganisms, including Bacillus subtilis spp.), protozoa (Sarcodina-amoeba, e.g., Entamoeba), and phages (e.g., Lactobacillus phage).

In embodiments, the methods according to the present invention are capable of extracting nucleic acid molecules from a variety of organisms, including fungi (i.e., Saccharomyces spp.), animal cells (Bos taurus), and plants (e.g., Hordeum vulgare).

The following examples are intended to illustrate, but not limit, the invention.

Extraction method A
10 mg to 5000 mg of sample was added to a sterile 2 milliliter (mL) microcentrifuge tube. Bead beating may be optionally performed by adding 400 microliters (μL) of the bead pure mixture and vortexing at 8000 rpm for about 30 seconds. However, if it is desired to obtain high molecular weight nucleic acids, such as genomic DNA, bead beating is preferably avoided.

First Lysate Treatment Step To lyse any Gram-negative bacteria in the sample, the sample was subjected to a first lysis solution by adding about 400 μL of digestion buffer (1% w/v SDS, 25 mM TrisHCl, 2.5 mM EDTA, 1% Triton™ X-100, pH 8) and about 20 μL of proteinase K to the sample and gently mixed. The mixture was then incubated at 55° C. for about 30 minutes.

Second Lysate Processing Step To lyse any Gram-positive bacteria in the sample, a second lysate containing glucoside hydrolase ("lysozyme") was added to the mixture resulting from the first lysate processing step to obtain a final lysozyme concentration of 1 mg/mL and a pH of about 8.0. Suitable glucoside hydrolases can be obtained from a variety of sources including egg whites, tears, or mucus or saliva of various animals. The mixture was then incubated at 37°C for about 1-24 hours.

Third Lysate Processing Step To lyse any fungal and/or yeast cells present in the sample, a third lysate containing 1 M lithium acetate and 5% w/v SDS in distilled, sterile H2O was added to obtain an approximately 1:5 dilution of the mixture resulting from the second lysate processing step. The processed mixture was incubated at 70°C for 15 minutes, followed by a heat shock at 95°C for 1 minute, and then brought to room temperature by placing in a 22°C water bath.

The second and third lysis solution treatment steps are sufficient to lyse the bacteriophage and viral coats, so no additional steps are required to extract genetic material from bacteriophages and viruses that may be present in the sample.

Extraction method B
Lysis Pretreatment Step: 100-200 mg of sample was added to a sterile 2 milliliter (mL) microcentrifuge tube. 500 mL of liquid nitrogen was added and the sample was frozen for 30 seconds. A pellet pestle or saw-tooth generator probe was then used to thoroughly grind the sample before proceeding to the next step.

First Lysate Treatment Step To lyse any animal, fungal, and protozoan food cell membranes in the sample, the sample was subjected to a first lysis solution by adding about 400 μL of digestion buffer (1% w/v SDS, 25 mM TrisHCl, 2.5 mM EDTA, 1% Triton™ X-100, 1.2 M NaCl pH 8) and about 20 μL of proteinase K to the sample and gently mixed. The mixture was then incubated at 55° C. for about 30 minutes.

Second Lysate Processing Step To lyse any fungal and/or yeast cells present in the sample, a second lysate containing 1 M lithium acetate and 5% w/v SDS in distilled, sterile H2O was added to obtain an approximately 1:5 dilution of the mixture resulting from the first lysate processing step. The processed mixture was incubated at 70°C for 15 minutes, followed by a heat shock at 95°C for 1 minute, and then brought to room temperature by placing in a 22°C water bath.

Nucleic Acid Purification In one embodiment, the genetic material extracted from the lysed microorganisms, i.e., the nucleic acid molecules present in the mixture after being subjected to the first, second, and third lysate processing steps, was then purified into DNA and RNA purification by splitting the mixture into two microcentrifuge tubes. DNA was extracted from one tube by adding about 20 μL of RNAse A and incubating at room temperature for 5 minutes. The mixture was passed through a biopolymer tissue homogenizer column. If bead-beating was performed beforehand, it is preferable to avoid passing the mixture through a tissue homogenizer column.

The eluate was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with approximately 400 μL of DNA lysis solution (guanidine HCl, Tris-EDTA, and 70% EtOH) and approximately 20 μL of proteinase K, mixed, and then incubated at 55°C for 10 minutes. EtOH was then added at -22°C and the mixture was mixed by inversion. The mixture may be subjected to one or more additional DNA extraction and purification methods known in the art.

RNA was extracted from the second microcentrifuge tube by passing the mixture through a biopolymer tissue homogenizer column. Again, if bead beating was performed beforehand, it is preferable to avoid passing the mixture through a tissue homogenizer column. The eluate was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with about 40 μL of DNase I (1 U) in 25 mM MgCl2 solution and then incubated at 37 degrees for about 15 minutes. The mixture was then subjected to acid guanidinium thiocyanate-phenol-chloroform extraction. The mixture may be subjected to one or more additional RNA extraction and purification methods known in the art.

In one embodiment, the genetic material extracted from the lysed microorganisms, i.e., the nucleic acid molecules present in the mixture after being subjected to the first, second, and pre-lysis treatment steps, was then DNA and RNA purified by splitting the mixture into two microcentrifuge tubes. DNA was extracted from one tube by adding approximately 20 L of RNAse A and incubating at room temperature for 5 minutes.

The eluate was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with approximately 400 μL of DNA lysis solution (guanidine HCl, Tris-EDTA, and 70% EtOH) and approximately 20 μL of proteinase K, mixed, and then incubated at 55°C for 10 minutes. EtOH was then added at -22°C and the mixture was mixed by inversion. The mixture may be subjected to one or more additional DNA extraction and purification methods known in the art.

The RNA was extracted from the second microcentrifuge tube. The eluate was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with about 40 μL of DNase I (1 U) in 25 mM MgCl2 solution and then incubated at 37 degrees for about 15 minutes. The mixture was then subjected to acid guanidinium thiocyanate-phenol-chloroform extraction. The mixture may be subjected to one or more additional RNA extraction and purification methods known in the art.

In some embodiments where quantitative expression of RNA molecules is desired, the use of RNA stabilizing buffers and bead beating is preferred to ensure release and limited degradation of the RNA nucleic acid molecules.

In some embodiments where extraction of high molecular weight nucleic acid molecules is desired, bead beating and tissue homogenization columns are avoided and phenol-chloroform-alcohol extraction is performed instead of silica column based extraction. In some embodiments, magnetic bead based nucleic acid purification may be performed. Agarose gel based purification and concentration may be performed to remove selective molecular weights of nucleic acids and purify the sample.

Metagenomics Analysis In one embodiment, extracted and purified genetic material was prepared for sequencing using Illumina index adapters to check size and quantity. Low cycle PCR was performed for 1-20 cycles for any input of less than 50 ng of DNA, otherwise PCR-free library preparation methods can be utilized for 50 ng or more of nucleic acid. Gel purification was performed using the Qiagen Gel Purification Kit™ (Qiagen, Frederick, MD). Clean PCR products were quantified using a Qubit™ 2.0 fluorometer (Life Technologies, Carlsbad, CA). Samples were combined in equimolar amounts. Library pools were size verified using a Fragment Analyzer™ CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using a Qubit™ High Sensitivity dsDNA kit (Life Technologies, Carlsbad, CA). After dilution and spiking with 1%-10% PhiX™ V3 Library Control (Illumina, San Diego CA), pools were denatured with an equal volume of 0.1 N NaOH for 5 minutes and then further diluted in Illumina's HT1 buffer. The denatured and PhiX™-spiked pools were loaded onto an Illumina Next Generation™ sequencer with Illumina sequencing primers set to 50-550 bases, paired end or single read.

This method can use sequencing reads in the range of 1000 or more for short insert methods. Large insert methods such as Pac Bio™, Nanopore™, or other next gene sequencing methods can use less than 1000 sequencing reads. Bioinformatics quality filtering was performed before taxonomy assignment. Quality trimming of raw sequencing files can include removing sequencing adapters or indexes; trimming the 3' or 5' ends of reads based on quality score (Q20>), terminal base pairs, or signal intensity; removing reads based on quality score, GC content, or unaligned base pairs; removing duplicate reads at a set number of base pairs. Alignment of processed sequencing files was performed using a custom microbial genome database consisting of sequences from refseq™, Greengeens™, HMP™, NCBI™, PATRIC™, or other public/proprietary data repositories or in-house datasets. This database can be used as a complete genome alignment scaffold, k-mer fragment alignment, or other schemes practiced in the art of metagenomics and bioinformatics. Organisms are assigned a common or unique taxonomic identity based on the number of sequencing reads/fragments that match the database genome. This identifier may be a barcode, nucleotide sequence, or some other computational tag that associates matching sequencing reads with organisms or strains within a taxon. Some identifiers are higher order and identify the domain, kingdom, phylum, class, order, family, or genus of the organism.

The present invention can identify organisms at the lowest order of strain within a species.

In an embodiment, the invention includes identification and/or analysis of one or more bacteria contained within our database (Figure 10). Some selected examples are Bacillus clausii, Bifidobacterium animalis, Pediococcus acidilactici, Acinetobacter indicus, Lactobacillus salivarius, Acinetobacter spp., Bacillus amyloliquefaciens, Lactobacillus helveticus, and others. helveticus, Bacillus subtilis, Lactobacillus plantarum, Bifidobacterium longum subsp. infantis, Enterococcus hirae, Lactobacillus delbrueckii subsp. bulgaricus, Enterococcus spp., Lactobacillus rhamnosus, Lactococcus lactis lactis, Pseudomonas stutzeri, Lactobacillus acidophilus, Klebsiella and Enterobacter cloacae strains.

In embodiments, the present invention includes the identification and/or characterization of one or more yeasts contained within our database (FIG. 10). Some selected examples are Saccharomyces species Boulardii, Saccharomyces kudriavzevii, Saccharomyces pastorianus, and Saccharomyces cerevisiae.

In embodiments, the invention includes the identification and/or analysis of one or more phages or viruses contained within our database (FIG. 10). Some selected examples are Bacillus phage phi29, Enterobacteria phage HK022, Lactobacillus phage A2, Escherichia phage HK639, phage cdtI, Sclerotinia sclerotiorum partitivirus S segment 2, Burkholderia phage BcepMu, Lactococcus prophage bIL311, Enterococcus phage phiFL4A, and Streptococcus phage SM1.

Future improvements to the database will increase or refine the organisms that can be detected using this method.

In one embodiment, the extracted and purified genetic material was prepared for sequencing using Illumina index adapters to check size and quantity. Low cycle PCR can be performed or standard PCR-free methods can be performed. Gel purification was performed using the Qiagen Gel Purification Kit™ (Qiagen, Frederick, MD). Clean PCR products were quantified using a Qubit™ 2.0 fluorometer (Life Technologies, Carlsbad, CA). Samples were combined in equimolar amounts. Library pools were size verified using a Fragment Analyzer™ CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using a Qubit™ High Sensitivity dsDNA kit (Life Technologies, Carlsbad, CA). After dilution and spiking with 10% PhiX™ V3 Library Control (Illumina, San Diego CA), pools were denatured with an equal volume of 0.1 N NaOH for 5 minutes and then further diluted in Illumina's HT1 buffer. The denatured and PhiX™ spiked pools were loaded onto an Illumina™ next-generation sequencer with Illumina sequencing primers set to 150 base paired-end reads. Bioinformatics quality filtering was performed prior to taxonomic assignment.

Using Table 1, determine that the individual has ingested:

(Table 1)

Monitoring macronutrient intake and dietary guidance In some embodiments, the present invention can be used to monitor the nutrition, quantity, and quality of food intake in a subject. For example, before treatment with probiotics, a sample obtained from the subject's digestive tract can be obtained, and the genetic material of the food organism therein can be extracted and subjected to metagenomics analysis as disclosed herein. A customized food-specific database consisting of whole, partial, or incomplete reference genomes, RNA, or nucleic acid components or fragments is utilized by bioinformatics tools to identify, quantify, and taxonomically assign nucleic acid information from sequencing. The output is exemplified in Table 2 below, and includes the identification of the species of organism or cells of the organism that were in the gut.

(Table 2)

Then, during and/or after treatment with a given probiotic, a second sample may be obtained from the subject's digestive tract and the genetic material of the microorganisms in the second sample may be extracted and subjected to metagenomic analysis, the results of which are compared to the results of the metagenomic analysis of the first sample. Based on the comparison results, the food biology results may then be compared to the microbiome biology results to understand the microorganisms associated with the food and to make an overall food quality assessment. In some embodiments, this may provide information about the species of organisms that an individual is ingesting through their food sources, and about any genetic modifications, mutations, or irregularities to that species, either by selection or direct modification.

In some embodiments, the second sample of the microbiome analysis allows for the detection of microorganisms common to the food organism and provides information about the health of the food organism. In some embodiments, food consumed by humans may be part of a common food source, such as chicken, cow, pig, or even plants and protists, whose species are identified and matched with their specific microorganisms. In certain exemplary embodiments, chicken species, which may have chicken sarcoma virus, may be detected in the analyzed second gut microbiome sample. In some embodiments, the health of the ingested food organism may be determined by the presence or absence of microorganisms that adversely affect the health of the host organism. In certain exemplary embodiments, it may be detected that diseases such as Equine Herpesvirus 2, a respiratory disease in horses, may affect the health of the host organism.

In some embodiments, the present invention can be used to screen the gut microbiome of a given subject and then custom tailor a food or dietary regimen to the subject that allows for improved health quality in terms of nutritional balance, improved microbial gut profile, and nutrient absorption.

Monitoring Probiotic Treatment In some embodiments, the present invention can be used to monitor probiotic treatment in a subject. For example, before treatment with a probiotic, a sample obtained from the subject's digestive tract can be obtained, and the genetic material of the microorganisms therein can be extracted and subjected to metagenomics analysis as disclosed herein. Then, during and/or after treatment with a given probiotic, a second sample can be obtained from the subject's digestive tract and the genetic material of the microorganisms in the second sample can be extracted and subjected to metagenomics analysis as disclosed herein, and the results are compared with the results of the metagenomics analysis of the first sample. Then, based on the comparison results, the subject's probiotic treatment can be modified to obtain a desired microbial population in the subject's intestine. For example, a probiotic containing a desired microorganism whose amount is increased in the subject's intestine can be administered to the subject.

In some embodiments, fecal samples may be mixed or cultured for determination of the metabolome of the microbial fecal population. The metabolomic profile may then be used to determine probiotic strains that will benefit the individual. Examples of metabolomic profiles include those that affect energy metabolism, nutrient utilization, insulin resistance, adiposity, dyslipidemia, inflammation, short chain fatty acids, organic acids, cytokines, neurotransmitters chemicals or phenotypes and may include other metabolomic markers.

Microbiome Screening and Probiotic Selection The present invention has been successfully used to determine the microbial content of various commercially available probiotics. In addition, the method of the present invention can be used to determine the microbial content of various probiotics and the microbiome content in the gut of a subject. In one embodiment, based on the microbiome content in the gut of a subject and any desired changes thereto, one or more probiotics can be selected that contain the microorganisms that are desired to be increased and/or maintained in the health of the microbiome of the subject. In one embodiment, based on the microbiome content in the gut of a subject and any desired changes thereto, one or more probiotics can be selected that contain the microorganisms that are desired to be increased and/or maintained in the gut balance of a subject in relation to the macronutrient content obtained from their food sources, recorded by direct survey information from the individual or by gut organism nucleic acid analysis according to the present invention.

Here, the microbiome represents the entirety of the microflora and the organisms contained therein, such as bacteria, fungi, viruses, phages, and parasites. For example, using the methods described herein, a subject's gut microbiome is determined to contain 25% A and 75% B, probiotic 1 is determined to contain 75% A and 25% B, and probiotic 2 is determined to contain 25% A and 75% B. If it is desired to maintain the subject's gut microbiome, probiotic 2 would be selected for administration to the subject. However, if it is desired that the amount of A and B in the subject's gut is 50/50, both probiotics 1 and 2 can be selected for administration to the subject. Alternatively, probiotic 1 may be selected for administration to the subject until the amount of A and B in the subject's gut reaches 50/50. In some embodiments, a probiotic formulation containing, for example, equal, variable, or varying amounts of A and B or other probiotic strains may be tailored to the subject for administration to the subject. Computational models utilizing the relative abundance of microbes present in an individual's gut can help determine the type, dose, and cocktail of microbes to include in the probiotic. For example, if organism A is determined to be reduced or absent compared to the general population or previous microbiome analysis, a probiotic or prebiotic is provided that increases the concentration of organism A. This prebiotic or probiotic can be just that organism A or another organism that supports the growth of organism A. The dose given will take into account the relative abundance of the organism in the individual, growth rate, fitness, receptors or receptor density, genes, or performance characteristics of the prebiotic/probiotic such as expression patterns, or metabolomic products.

The subject-tailored probiotics are formulated based on the relative abundance detected in individual gut/fecal samples, although not necessarily in equal amounts. These formulations are made to modulate the microbiome to a healthy state. The health of the microbiome is determined by the use of existing aggregate private and public databases such as metaHIT™, Human Microbiome Project™, American Gut Project™, etc. Health status may be determined individually for those who are healthy with no known issues in terms of blood biomarker testing, after which their complete microbiome profile is completed. After one or several microbiome signatures are completed, the average of some/all of the microbes found can be understood for that individual, and the variance from that average can be accessed to determine if they are in dysbiosis. The microbiome profiles can be aggregated into groups, and these groups are then assigned barcodes for rapid bioinformatics assignment. Groups can be created by single or multiple phenotypic, diagnostic, or demographic information associated with the individual from whom the sample was collected. Unique groups can be determined from other groups by comparison to aggregated known populations, such as "normal" as defined by the Human Microbiome Project or the American Gut Project, using linear distance calculations, diversity values, classifiers such as C4.5 decision trees, or statistical models such as principal component analysis.

Thus, in some embodiments, the present invention can be used to screen the gut microbiome of a given subject and then tailor a probiotic regimen to the given subject based on the subject's gut microbiome.

Treatment of Dysbiosis In some embodiments, the present invention may be used to restore a subject's gut flora and/or fauna to homeostasis following an event in which the subject's microbiome has transitioned from a balanced microbiome to one that is causing or may cause negative side effects, disorders, and/or disease. Health conditions include, but are not limited to, a variety of conditions, from acne and allergies to gastrointestinal disorders, obesity, and cancer. One example of such a dysbiosis is the onset of obesity. It has been shown that some microbial strains in a subject's gut are associated with obesity or weight management issues that the subject is subject to. See, e.g., Ley, et al. (2005) PNAS USA 102:11070-11075. For example, in obese animals and human subjects, the ratio of Bacterides to Firmicutes microbes plays an important role in metabolic performance. See, e.g., Turnbaugh, et al. (2012) PLOS ONE 7:e41079. Some gut microbes known to be associated with obesity and weight control issues include Bacteroides uniformis, Bacteroides pectinophilus, Roseburia inulinivorans, Methanobrevibacter smithii, and Bifidobacterium animalis.

Thus, in some embodiments, the ratio of a first given microorganism to a second given microorganism in a subject's gut is determined using the methods described herein, and then, if the ratio is undesirable or abnormal, the subject is administered a treatment to correct the ratio to a desired ratio. In some embodiments, the amount of a first given microorganism in a subject's gut relative to the total amount of all microorganisms in the subject's gut is determined using the methods described herein, and then, if the relative amount of the first given microorganism is undesirable or abnormal, the subject is administered a treatment to correct the amount to a desired amount. Retesting of the gut microbiome may be used to determine whether they are adhering well to macronutrient and dietary recommendations. Such treatments include administering to the subject: probiotics containing one or more microorganisms whose abundance is desired to be increased in the subject's gut, antimicrobial agents such as antibiotics, antifungals, antivirals, etc., to kill or slow the growth of a microorganism or microorganisms whose abundance is desired to be reduced in the subject's gut, diet and/or nutritional supplements that support the growth or maintenance of a healthy gut microbiome, such as prebiotics, magnesium, fish oil, L-glutamine, vitamin D, etc. For example, Million, et al. ((2005) Int. J. Obes. 36:817-825) show that the gut microbiota of obese subjects is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii. Thus, after using the methods described herein to determine the amount of Lactobacillus reuteri, Bifidobacterium animalis, and Methanobrevibacter sumidii in a subject's gut and finding that the amount is typical or indicative of an obesity-associated gut microbiome, the subject can be administered a probiotic that contains Bifidobacterium animalis and Methanobrevibacter sumidii and relatively little to no Lactobacillus reuteri. In embodiments, the gut microbiome of an obese subject would benefit from a diet having flavonoids, polyphenols, and short chain fatty acids.

Scoring the Microbiome Scoring the overall microbiome signature uses similar decision trees, algorithms, artificial intelligence, scripts, or logic trees, as shown in Table 3. This system allows for a score that helps the user understand how healthy their gut microbiome is and whether they need to address some or many of the challenges found. Challenges may include, but are not limited to, identification of known pathogenic organisms, number and identification of opportunistic pathogens, potential organisms known to cause pathogenic effects when given the opportunity, lack of support for a good microbial environment but their composition or lack of major strains, overall diversity, and number of unique organisms found in the top 10, and/or organisms with a prevalence greater than 0.1%.

A diversity cut off was determined from the aggregate of sample analysis, and the cut off was determined at x relative abundance. For example, if x=0.1%, then 352 unique organisms constitute an average healthy profile. Then, by applying standard deviation around this number, and using Gaussian distribution and percentiles under curve analysis, we can score how close the average diversity number is from the database mean. The lower the diversity number and the further away from the mean, the lower the microbiome score. The higher the number and the greater the diversity, the higher the microbiome score. This type of scoring category along with the probiotic score determines a number and visually measured score for customization to understand how healthy the microbiome is. An example of a graphic visualization is shown below. Low means poor microbiome quality, high means high microbiome quality and score. Low->30 out of 100, Medium>65 out of 100, High=65 or more out of 100.

An example of a scoring and probiotic formula algorithm is included in Table 3 below. Table 3 may be represented as a decision tree, algorithm, artificial intelligence, script, or logic tree. Such a decision tree, algorithm, artificial intelligence, script, or logic tree function outputs an individual microbiome wellness score associated with the detected probiotics and provides formula and dosing recommendations for probiotic use.

An exemplary list of possible categories into which microorganisms may be grouped is provided below in Table 4.

Table 3: Example of a probiotic scoring and formula determination table, including use of a probiotic strain database, a metagenomics database, and a literature curation database.

(Table 4) Possible categories for grouping

All scientific and technical terms used in this application have the meaning commonly used in the art unless otherwise specified.

As used herein, the term "subject" includes humans and non-human animals. The term "non-human animals" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects and test animals.

The use of the singular can include the plural unless specifically stated otherwise. As used herein and in the appended claims, the singular forms "a," "an," and "the" can include plural referents unless the context clearly dictates otherwise. The use of "or" can mean "and/or" unless specifically stated otherwise. As used herein, "and/or" means "and" or "or." For example, "A and/or B" means "A, B, or both A and B," and "A, B, C, and/or D" means "A, B, C, D, or combinations thereof," where "combinations thereof" refers to any subset of A, B, C, and D, such as a single member subset (e.g., A or B or C or D), a two-member subset (e.g., A and B, A and C, etc.), or a three-member subset (e.g., A, B, and C, or A, B, and D, etc.), or all four members (e.g., A, B, C, and D).

As used herein, the terms "sample" and "biological sample" refer to any sample suitable for the methods provided by the present invention. A sample of cells can be any sample, including, for example, an intestinal or fecal sample obtained by non-invasive or invasive techniques such as a biopsy of a subject. In one embodiment, the term "sample" refers to any preparation derived from a subject's feces or intestinal tissue. For example, a sample of cells obtained using the non-invasive methods described herein can be used to isolate nucleic acid molecules or proteins for the methods of the present invention.

In embodiments, the analysis can be of any nucleic acid, including DNA, RNA, cDNA, miRNA, mtDNA, single-stranded or double-stranded. The nucleic acid can be of any length, from as short as about 5 bp oligos to as long as megabases or longer. As used herein, the term "nucleic acid molecule" refers to DNA, RNA, single-stranded, double-stranded or triple-stranded, as well as any chemical modifications of these. Virtually any modification of a nucleic acid is contemplated. "Nucleic acid molecules" include 10, 20, 30, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, It may be 15,000, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, 200,000, 500,000, 1,000,000, 1,500,000, 2,000,000, 5,000,000 or more bases in length, and can be almost any length up to a full-length chromosomal DNA molecule. For methods of analyzing gene expression, the nucleic acid isolated from the sample is usually RNA.

A single-stranded nucleic acid molecule is "complementary" to another single-stranded nucleic acid molecule if it can base pair (hybridize) with all or a portion of the other nucleic acid molecule to form a double helix (double-stranded nucleic acid molecule) based on the ability of guanine (G) to base pair with cytosine (C) and adenine (A) to base pair with thymine (T) or uridine (U). For example, the nucleotide sequence 5'-TATAC-3' is complementary to the nucleotide sequence 5'-GTATA-3'.

As used herein, "hybridization" refers to the process by which a nucleic acid strand binds to a complementary strand through base pairing. Because the hybridization reaction is sensitive and selective, a particular sequence of interest can be identified even in samples in which it is present at low concentrations. In an in vitro situation, suitably stringent conditions can be defined, for example, by the concentration of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. In particular, stringency can be increased by decreasing the concentration of salt, increasing the concentration of formamide, or increasing the hybridization temperature. For example, hybridization under high stringency conditions can occur in about 50% formamide at about 37°C to 42°C. Hybridization can occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C. In particular, hybridization can occur under high stringency conditions at 42° C. in 50% formamide, 5x SSPE, 0.3% SDS, and 200 mg/ml sheared and denatured salmon sperm DNA. Hybridization can occur under reduced stringency conditions as described above, but in 35% formamide at a reduced temperature of 35° C. The temperature range corresponding to a particular stringency level can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.

As used herein, the term "microbiome" refers to the microorganisms, including bacteria, viruses, and fungi, archaea, protozoa, amoebas, or helminths, that inhabit the gut of a subject.

As used herein, the terms microbial, microbe, or microorganism refer to any microorganism, including prokaryotes or eukaryotes, spores, bacteria, archaea, fungi, viruses, or protozoans, unicellular or multicellular.

The present invention is described in part in terms of functional components and various processing steps. Such functional components and processing steps may be realized by any number of components, operations, and techniques configured to perform the specified functions and achieve various results. For example, the present invention may employ a variety of biological samples, biomarkers, elements, materials, computers, data sources, storage systems and media, information collection techniques and processes, data processing standards, statistical analyses, regression analyses, and the like, which may perform various functions. In addition, although the present invention is described in the context of medical diagnostics, the present invention may be implemented in conjunction with any number of applications, environments, and data analyses, and the systems described herein are merely exemplary applications for the present invention.

The methods for data analysis according to various aspects of the invention may be implemented in any suitable manner, for example, using a computer program running on a computer system. An exemplary analysis system according to various aspects of the invention may be implemented in conjunction with a computer system, for example, a conventional computer system including a processor and random access memory, such as a remotely accessible application server, network server, personal computer or workstation, etc. The computer system also preferably includes additional memory devices or information storage systems, such as a mass storage system, and a user interface, such as a conventional monitor, keyboard, and tracking devices. However, the computer system may include any suitable computer system and associated equipment and may be configured in any suitable manner. In one embodiment, the computer system includes a stand-alone system. In another embodiment, the computer system is part of a network of computers including servers and databases.

The software necessary to receive, process, and analyze the genetic information may be implemented on a single device or on multiple devices. The software may be accessible over a network such that storage and processing of the information is performed remotely with respect to the user. The analytical system and its various elements according to various aspects of the present invention provide functions and operations to facilitate microbiome analysis, such as data collection, processing, analysis, reporting, and/or diagnosis. The analytical system maintains information about the microbiome and samples to facilitate analysis and/or diagnosis. For example, in the present embodiment, the computer system executes a computer program that may receive, store, retrieve, analyze, and report information about the microbiome. The computer program may include multiple modules that perform various functions or operations, such as a processing module for processing raw data and generating supplemental data, and an analytical module for analyzing the raw data and supplemental data to generate models and/or predictions.

The analysis system may also provide various additional modules and/or individual functions. For example, the analysis system may also include a reporting function to provide information related to the processing and analysis functions, for example. The analysis system may also provide various administrative and operational functions, such as controlling access and performing other administrative functions.

To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference to the same extent as if each were individually incorporated as such.

Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims (1)

a) mixing the sample with a liquid nitrogen solution;
b) adding a first solution, the first solution including a surfactant and a chelating agent;
and c) adding a second lysis solution, said second lysis solution comprising a chaotropic agent.

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