CA3095745A1 - Universal method for extracting nucleic acid molecules from a diverse population of one or more types of microbes in a sample - Google Patents

Abstract

Disclosed herein are methods of extracting genetic material from a diverse population of one or more types of microbes in a sample. Microbes can be prokaryotes or eukaryotes 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 through a molecular method such as qPCR, PCR, RFLP, SSCP, allele specific PCR, targeted sequencing, pull down sequencing, whole shotgun sequencing, or other methods. Also provided are methods that include extracting nucleic acid molecules from a variety of organisms such as fungi (i.e., Saccharomyces spp.), animal cells (Bos taurus), plants (e.g., Hordeum vulgare) from the gut of a human subject, performing a metagenomics analysis therefrom, and determining a probiotic treatment or dietary guidance for the subject based on the metagenomics analysis.

Description

2 UNIVERSAL METHOD FOR EXTRACTING
NUCLEIC ACID MOLECULES FROM A DIVERSE POPULATION OF
ONE OR MORE TYPES OF MICROBES IN A SAMPLE
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 119(e) to U.S.
Application Serial No. 62/651,620 filed April 2, 2018. The disclosure of the prior application is considered part of and is incorporated by reference in the disclosure of this application.
BACKGROUND OF THE INVENTION
FIELD OF INVENTION
[0002] The present invention relates generally to genomic analysis and more particularly to a method of extracting and analyzing nucleic acid molecules associated with food from a diverse population of microbes in a biological sample.
BACKGROUND INFORMATION

[0003] About 100 trillion microorganisms live in and on the human body vastly outnumbering the body's approximately 10 trillion human cells. These normally harmless viruses, bacteria and fungi are referred to as commensal or mutualistic organisms.
Commensal and mutualistic organisms help keep our bodies healthy in many ways.

Together all of the microorganisms living in and on the body¨commensal, mutualistic and pathogenic¨are referred to as the microbiome and their equilibrium and associated metabolome is closely linked to an individual's health status and vice-versa.

[0004] Advances in nucleic acid sequencing has created an opportunity to quickly and accurately identify and profile the microbiome inhabiting the gut and subcutaneous tissue.
The optimal flora also interacts with the host immune system in a synergistic way further propagating its health benefits. The associated metabolome of individuals can also be profiled either by a mass-spectrometry based system or using genomics-based metabolome modeling and flux-balance analysis and used to make a healthy metabolome profile. All these methodologies can be used to dissect the complexity of microbial communities.

SUMMARY OF THE INVENTION

[0005] The present invention is directed to a method of extracting nucleic acid molecules from a diverse population of microbes in a biological, environmental, dietary supplement, or other ecological microbial organism heterogeneous populations sample and use of nucleic acid or extracts through processing steps and analysis for the determination of probiotic customization to an individual. Processing steps specific to this invention include, RNA or DNA clean-up, fragmentation, separation, or digestion; library or nucleic acid preparation for downstream applications, such as PCR, qPCR, digital PCR, or sequencing; preprocessing for bioinformatic QC, filtering, alignment, or data segregation;
metagenomics or human genomic bioinformatics pipeline for microbial species taxonomic assignment; and other organism alignment, identification, and variant interpretation.

[0006] The present invention also describes a universal method for using samples for DNA extraction and determination of food consumption based on food DNA
sequence from a database of meats, plants, fruits, vegetables, and/or microbes contained with these organisms. Disclosed herein are methods of extracting genetic material from a diverse population of one or more types of cells or cell components in a sample and determining the consumed food and nutritional breakdown for the improvement of health and prevention of disease.

[0007] Accoridingly, in one aspect, the invention provides a method for 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 chelator, e.g., EDTA; b) adding a second lysis solution having a 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, and the like, to the mixture of step b). Pre-processing steps may include physical lysis may be used to further optimize nucleic acid yield. Examples of mechanical lysis include sonication, bead mixing, and bead mill homogenization.

[0008] 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 comprising a detergent and a chelator, e.g., SDS, and a chelator, e.g., EDTA; and c) adding a second lysis solution, the second lysis solinion including a chaotropic agent, e.g., urea, lithium acetate, guanidine hydrochloride. Pre-processing steps may include physical lysis may be used to further optimize nucleic acid yield. Examples of mechanical lysis include sonication, bead mixing, and bead mill homogenization.

[0009] In another aspect, the invention provides a method of determining food consumption of a subject. The method includes: a) extracting genetic material from a stool sample obtained from the subject, said genetic material extracted according to a method of the disclosure; and b) subjecting the genetic material extracted from the first sample to metagenomics analysis to determine the food consumption of the subject. In embodiments, the method further includes treating the subject with a probiotic or a food stuff based on the analysis of food consumption.

[0010] In another aspect, the invention provides a method of monitoring a probiotic treatment of a subject. The method includes: a) extracting genetic material from any microbes present in a first sample obtained from the subject, said genetic material extracted according to a method of the disclosure; 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 microbes present in a second sample obtained from the subject in the same manner as the extraction of genetic material from the first sample; d) performing metagenomics analysis on the extracted genetic material from the second sample; and e) comparing the results of the metagenomics analysis of the first sample with the metagenomics analysis of the second sample.

[0011] In yet another aspect, the invention provides a method comprising calculating a probiotic score from probiotic organisms detected in a gut with or without additional chemistry or genetic tests.

[0012] In still another aspect, the invention provides a method comprising calculating a score for a microbiome, the score being used to assess if the microbiome is in dysbiosis, neutral, or stable.

[0013] The invention further provides a computing system comprising: a memory; and one or more processors coupled to the memory, the one or more processors configured to perform operations to perform a method of the present invention.

[0014] The invention also provides an automated platform for performing a method of the invention.

[0015] The invention provides an all-in-one method for extracting nucleic acids from a diverse flora of microbes from a biological, environmental, dietary supplement, or other ecological microbial organism heterogeneous populations sample.

[0016] In embodiments, the invention may be used in determining composition and relative abundance of microbes, via analyzing their respective nucleic acids, in probiotics and environmental samples. DNA is purified and used downstream for nucleic acid analysis (notably metagenomics analysis where genome of more than one species/subspecies is identified).

[0017] 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 and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram illustrating the presence of high prevalence organisms of a microbiome signature of a human (high protein diet, >50 years old, supplement user).

[0019] Figure 2A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).

[0020] Figure 2B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).

[0021] Figure 2C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).

[0022] Figure 2D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, vegetarian diet).

[0023] Figure 3A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non-vegetarian diet).

[0024] Figure 3B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non-vegetarian diet).

[0025] Figure 3C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non-vegetarian diet).

[0026] Figure 3D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high carbohydrate diet, 18-50 years old, non-vegetarian diet).

[0027] Figure 4A is a schematic diagram illustrating the presence of high prevalence organisms (bacteria) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).

[0028] Figure 4B is a schematic diagram illustrating the presence of high prevalence organisms (viruses and phages) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).

[0029] Figure 4C is a schematic diagram illustrating the presence of high prevalence organisms (archaea) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).

[0030] Figure 4D is a schematic diagram illustrating the presence of high prevalence organisms (fungi and other eukaryotes) of a microbiome signature of a human (high dairy protein diet, 0-2 years old, vegetarian non-nursing).

[0031] Figure 5 is a schematic diagram illustrating the presence lower prevalent organisms and identification of opportunistic pathogens of a microbiome signature of a human.

[0032] Figure 6 is a schematic diagram illustrating typical probiotics detected in a microbiome signature of a human.

[0033] Figure 7 is a schematic diagram illustrating typical probiotics detected in a microbiome signature of a human.

[0034] Figure 8 is a schematic graphical plat illustrating showing comparison of individual relative abundance to database average for normal population.

[0035] Figure 9 is a table setting forth organisms identified via the method of the invention from a dietary supplement mixed culture.

[0036] Figure 10 is a table setting forth the classification of unique species of various microbes stored in the database of the invention.

[0037] Figure 11 illustrates example demographic information from an individual in one embodiment of the invention.

[0038] Figure 12 illustrates example organisms detected related to seafood in one embodiment of the invention.

[0039] Figure 13 illustrates example organisms detected related to mammalian meats in one embodiment of the invention.

[0040] Figure 14 illustrates example organisms detected related to grains in one embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides a universal method for extracting nucleic acid molecules from a diverse population of one or more types of microbes in a sample. The types of microbes include: gram-positive bacteria, gram-positive bacterial spores, gram-negative bacteria, archaea, protozoa, helminths, algae, fungi, fungal spores, viruses, viroids, bacteriophages, and rotifers. In some embodiments, the diverse population is a plurality of different microbes of the same type, e.g., gram-positive bacteria. In some embodiments, the diverse population is a plurality of different types of microbes, e.g., bacteria (gram-positive bacteria, gram-positive bacterial spores and/or gram-negative), fungi, viruses, and bacteriophages.

[0042] Because different types of microbes have different compositions and mechanisms to protect their own genetic material it is often difficult to extract the genetic material from one type of microbe without compromising the ability to also extract the genetic material of another type of microbe in the same biological sample. The present invention, however, allows the extraction of genetic material from different types of microbes in a sample without sacrificing the amount of genetic material that can be obtained from one type of microbe by extracting the genetic material of another type of microbe in the same sample. According to the present invention, the sample comprising the microbes may be a biological sample, environmental sample, an artificially created sample (e.g., a laboratory test or control sample, a sample of a probiotic composition or supplement, etc.), or the like. 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.), and the like. Solid samples may be liquefied or mixed with a solution, and then genetic material of the microbes present in the liquefied sample, mixture, or solution obtained from the mixture may be extracted in accordance with the present invention. The extracted genetic material may be subjected to further processing and analysis such as purification, amplification, and sequencing.

[0043] In some embodiments, the extracted genetic material is subjected to metagenomics analysis to, for example, identify the one or more types of microbes in the sample from which the genetic material was extracted. In additional embodiments, full whole genome shotgun sequencing can be performed on prepared extracted nucleic acid material from human fecal samples. Preparations include nucleic acid clean up reactions to remove organic solvents, impurities, salts, phenols, and other process inhibiting contaminants. Additional preparations include nucleic acid library prep from each sample where the gDNA is subject to modifications and/or amplifications to prep the sample for sequencing on a sequencing platform such as massively parallel sequencing by synthesis, nanopore, long read, and/or CMOS electronic, sequencing methods.

[0044] As disclosed herein, the inventive method allows the successful extraction of genetic material from one or more different types of microbes present in the same sample by subjecting the microbes to three different compositions in a particular order. The method according to the present invention comprises first lysing any gram-negative bacteria present in the sample, which is followed by digesting the polysaccharide component of the cell walls of any yeast and bacteria present in the sample, and then disrupting any cell walls that are intact after the second step with a chaotropic agent.

[0045] Briefly, in an embodiment, the first step comprises mixing the sample with a first lysis solution comprising a detergent (e.g., sodium dodecyl sulfate (SDS)) and a chelator (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 Im X-100), and/or one or more proteases (e.g., proteinase K).

[0046] After the first step, the sample is mixed with a second lysis solution comprising a lysozyme to digest the polysaccharide component of any yeast and bacterial cell walls present in the mixture. Because lysozyme may inhibit the activity of the first lysis solution, it is important that contact of the sample with the second lysis solution occurs after treating the sample with the first lysis solution.

[0047] After treatment with the second lysis solution, a third lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, and the like) is added to the mixture to disrupt any cell walls that are not digested by the second lysis solution. The third lysis solution may include a detergent such as SDS.

[0048] In some embodiments, both the first lysis solution and the third lysis solution comprise SDS at a working concentration of between 1-10% w/v. In some embodiments, after treatment with the third lysis solution, the mixture is further treated with a fourth lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, and the like) and Proteinase K. In some embodiments where the chaotropic agent of the third lysis solution is lithium acetate, the mixture is then subjected to heat shock treatment and may then be treated with the fourth lysis solution.

[0049] In certain aspects, the following disclosure describes a universal method for using stool samples for DNA extraction and determination of food consumption based on food DNA sequence from a database of meats, plants, fruits, vegetables, and/or microbes contained with these organisms. Disclosed herein are methods of extracting genetic material from a diverse population of one or more types of cells or cell components in a sample and determining the consumed food and nutritional breakdown for the improvement of health and prevention of disease.

[0050] In some embodiments, biological secretions (e.g., semen, vaginal secretions, breast milk, tears, saliva, blood, urine, and the like) are obtained from the digestive tract, and the like. Solid samples may be liquefied or mixed with a solution, and then genetic material of any food item containing genetic material, such as plant based (seedlings, leaves, cotyledons, seeds, endosperm, tissue culture callus, roots, and the like), animal based, fungi based, or protista based foods in the liquefied sample, mixture, or solution obtained from the mixture may be extracted in accordance with 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 to, for example, identify the one or more types of organisms in the sample from which the genetic material was extracted.

[0051] In some embodiments the database that the metagenomic analysis will utilize has been customized for a specific purpose of identifying and taxonomically assigning, within the appropriate phylogeny, the nucleic acids with relative abundances of organisms or components of organisms ingested by humans or other animals. In some embodiments and additional data table or database may be used as a lookup of the relative abundances of organisms to determine macronutrient content of an organism's gut sample as a representation of their diet. In some embodiments this macronutrient breakdown may include fats, carbohydrates, proteins, vitamins minerals, and subcomponents of any macronutrients.

[0052] As disclosed herein, the inventive method allows the successful extraction of genetic material from one or more different types of organisms, one or more of an organism's cells, or cellular matrices or organelles present in the same sample by subjecting the sample to isolation, purification, or other methods for capturing nucleic acids. The method according to the present invention comprises lysing or disrupting any food cells in the sample, including but not limited to any cell walls and cell membranes, digesting the polysaccharide or lignin component of any cell walls or membranes of any fungi, plant, mammalian, or protista cells present in the sample, and disrupting any cell walls that are intact after the digestion step with a chaotropic agent.

[0053] The present invention includes a step to physically disrupt the cell wall or membranes of food cells by liquid nitrogen flash freezing and immediate mechanical disruption or grinding to break down cell walls and keep harmful cell enzyme inactivated prior to chemical lysis. The present invention includes a step comprising mixing the sample with a first lysis solution comprising a detergent (e.g., sodium dodecyl sulfate (SDS)) and a chelator (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 im X-100, Cetyltrimethylammonium bromide), and/or one or more proteases (e.g., proteinase K). In particular embodiments, the first lysis solution comprises SDS at a working concentration 1-10% w/v. The present invention includes a step comprising mixing the sample with a second lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, and the like). The second lysis solution may include a detergent, such as SDS. In particular example embodiments, the first and second lysis solutions may be added in any particular order.

[0054] In some embodiments, the present invention may include a step comprising mixing the sample with a third lysis solution comprising a lysozyme to digest the polysaccharide component of any fungi or bacteria cell walls present in the mixture. In some embodiments, the mixture may be further treated with a fourth lysis solution comprising a chaotropic agent (e.g., urea, lithium acetate, guanidine hydrochloride, and the like) 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 heat shock treatment and may then be treated with the fourth lysis solution. In particular example embodiments, the third and/or fourth solution may be added to the mixture at any point to disrupt any cell walls that are not digested by any previous lysis solution.

[0055] 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 that induces germination of the cell walls of the spores before contact with the first lysis solution. The pretreatment step may comprise mixing the sample with a chemical such as a mild detergent, e.g., Tween-80, to induce germination or cultivating the sample under conditions (e.g., temperature) that induce germination. In some embodiments, where germination is induced with a chemical, the chemical is preferably one that does not inhibit, reduce, or modify the activity or effectiveness of the first, second, and third lysis solutions.

[0056] In some embodiments, the method according to the present invention may further include one or more mechanical treatment steps that cause physical lysis by mechanical methods including sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, and the like. In some embodiments, a mechanical treatment step is performed before subjecting the sample to the first lysis solution.

[0057] In embodiments, the method according to the present invention is capable of extracting nucleic acid molecules from a variety of microbes including 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 (tapeworm Echinococcus spp.), Protozoa (Sarcodina ¨ the ameba, e.g., Entamoeba) and phages (e.g., Lactobacillus phages).

[0058] In embodiments, the method according to the present invention is capable of extracting nucleic acid molecules from a variety of organisms including fungi (i.e., Saccharomyces spp.), animal cells (Bos taurus), plants (e.g., Hon:leum vuigare).

[0059] The following examples are intended to illustrate but not to limit the invention.
EXTRACTION METHOD A

[0060] A range of 10mg to 5000mg of sample were added to a sterile 2 milliliters (mL) micro centrifuge tube. Bead beating may optionally be performed by adding 400 microliters (4) of bead pure mixture and vortexing for about 30 seconds at 8000 rpm. If, however, high-molecular weight nucleic acids, e.g., genomic DNA, are desired to be obtained, bead beating is preferably avoided.
First Lysis Solution Treatment Step

[0061] To lyse any gram-negative bacteria in the sample, the sample was subjected to a First Lysis Solution by adding about 400 uL of Digestion Buffer (1% w/v SDS, 25 mM
Tris HC1, 2.5 mM EDTA, 1% TritonTm X-100, pH 8) and about 20 uL of Proteinase K to the sample and gently mixed. The mixture was then incubated for about 30 minutes at 55 C.

Second Lysis Solution Treatment Step

[0062] To lyse any gram-positive bacteria in the sample, a Second Lysis Solution comprising a glucoside hydrolase ("lysozyme") was added to the mixture obtained from the First Lysis Solution Treatment Step to give a final lysozyme concentration of 1 mg/mL
and a pH of about 8Ø Suitable glucoside hydrolases may be obtained from a variety of sources including egg whites, tears, or mucus or saliva of various animals.
The mixture was then incubated for a period of about 1 to 24 hours at 37 C.
Third Lysis Solution Treatment Step

[0063] To lyse any fungal and/or yeast cells present in the sample, a Third Lysis Solution comprising 1M lithium acetate in distilled sterile H20 and 5% w/v SDS
was added to obtain about a 1:5 dilution of the mixture resulting from the Second Lysis Solution Treatment Step. The treated mixture was incubated for 15 minutes at followed by heat shock at 95 C for one minute and then brought to room temperature by placing in a 22 C water bath.

[0064] As the Second and Third Lysis Solution Treatment Steps are sufficient to lyse the outer coats of bacteriophages and viruses, no additional step is needed for extracting the genetic material from bacteriophages and viruses that may be present in the sample.
EXTRACTION METHOD B
Pre-Lysis Treatment Step

[0065] 100-200 mg of sample were added to a sterile 2 milliliters (mL) micro centrifuge tube. Add 500mL of liquid nitrogen and allow sample to freeze for 30sec.
Then using a pellet pestle or saw-tooth generator probe, grind the sample thoroughly before continuing to the next step.
First Lysis Solution Treatment Step

[0066] To lyse any animal, fungi, and protista food cell membranes in the sample, the sample was subjected to a First Lysis Solution by adding about 400 [EL of Digestion Buffer (1% w/v SDS, 25 mM Tris HC1, 2.5 mM EDTA, 1% Triton Tm X-100, 1.2M NaCl pH 8) and about 20 [EL of Proteinase K to the sample and gently mixed. The mixture was then incubated for about 30 minutes at 55 C.

Second Lysis Solution Treatment Step

[0067] To lyse any fungal and/or yeast cells present in the sample, a second Lysis Solution comprising 1M lithium acetate in distilled sterile H20 and 5% w/v SDS
was added to obtain about a 1:5 dilution of the mixture resulting from the first lysis solution treatment step. The treated mixture was incubated for 15 minutes at 70 C
followed by heat shock at 95 C for one minute and then brought to room temperature by placing in a 22 C water bath.
NUCLEIC ACID PURIFICATION

[0068] In an embodiment, the genetic material extracted from the lysed microbes, i.e., the nucleic acid molecules present in the mixture after being subjected to the First, Second, and Third Lysis Solution Treatment Steps were then purified to DNA and RNA
purification by splitting the mixture into two microcentifuge tubes. DNA was extracted from one tube by adding about 20 IA RNAse A and incubating for 5 minutes at room temperature. The mixture was run through a biopolymer tissue homogenizer column. If bead beating was previously performed, subjecting the mixture to the tissue homogenizer column is preferably avoided.

[0069] The eluate was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with about 400 uL of DNA Lysis Solution (Guanidine HC1, Tris-EDTA, and

70%
Et0H) and about 20 IA of Proteinase K, mixed, and then incubated at 55 C for minutes. Then Et0H at -22 C was added and the mixture was mixed by inverting.
The mixture may be subjected to one or more additional DNA extraction and purification methods known in the art.
[0070] RNA was extracted from the second microcentrifuge tube by running the mixture through a biopolymer tissue homogenizer column. Again, if bead beating was previously performed, subjecting the mixture to the tissue homogenizer column is preferably avoided. The eluate was then centrifuged at 1000 g for 5 minutes.
The supernatant was treated with about 40 IA DNase I (1 U) in a solution of 25 mM
MgCl2 and then incubated at 37 for about 15 minutes. Then the mixture was 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.

[0071] In an embodiment, the genetic material extracted from the lysed microbes, i.e., the nucleic acid molecules present in the mixture after being subjected to the First, Second, and pre lysis Treatment Steps were then purified to DNA and RNA
purification by splitting the mixture into two microcentifuge tubes. DNA was extracted from one tube by adding about 20 ?L RNAse A and incubating for 5 minutes at room temperature.

[0072] The eluent was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with about 400 uL of DNA Lysis Solution (Guanidine HC1, Tris-EDTA, and 70%
Et0H) and about 20 IA of Proteinase K, mixed, and then incubated at 55 C for minutes. Then Et0H at -22 C was added and the mixture was mixed by inverting.
The mixture may be subjected to one or more additional DNA extraction and purification methods known in the art.

[0073] RNA was extracted from the second microcentrifuge tube. The eluent was then centrifuged at 1000 g for 5 minutes. The supernatant was treated with about 40 uL DNase 1(1 U) in a solution of 25 mM MgCl2 and then incubated at 37 for about 15 minutes.
Then the mixture was 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.

[0074] In some embodiments, where the quantitative expression of RNA
molecules is desired, the use of an RNA stabilization buffer and bead beating is preferred to ensure release and limited degradation of RNA nucleic acid molecules.

[0075] In some embodiments where extraction of high molecular weight nucleic acid molecules is desired, bead beating and tissue homogenization column are avoided and phenol-chloroform-alcohol extraction is performed instead of silica column based extraction. In some embodiments a magnetic bead based nucleic acid purification may be performed. To remove selective molecular weights of nucleic acids and purify the sample, an agarose gel based purification and enrichment may be performed.

ME TAGENOMICS ANALYSIS

[0076] In an embodiment, the extracted and purified genetic material was prepared for sequencing using Illumina index adaptors and checked for sizing and quantity.
Low cycle PCR was performed between 1-20 cycles for any input less then 5Ong of DNA, otherwise PCR-Free methods of library prep can be utilized for 5Ong of nucleic acid or greater. Gel purification was performed using the Qiagen Gel Purification KitTm (Qiagen, Frederick, MD). Clean PCR products were quantified using the Qubiem 2.0 Fluorometer (Life Technologies, Carlsbad, CA). Samples were combined in equimolar amounts.
Library pools were size verified using the Fragment AnalyzerTm CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using the QubitTm High Sensitivity dsDNA
kit (Life Technologies, Carlsbad, CA). After dilution, a 1% to 10% spike of PhiXTm V3 library control (Illumina, San Diego CA), pools were denatured for 5 minutes in an equal volume of 0.1 N NaOH then further diluted in Illumina's HT1 buffer. The denatured and PhiXTm-spiked pool was loaded on an Illumina Next GenerationTm Sequencer with Illumina sequencing primers and set for between 50 - 550 base, paired-end or single reads.

[0077] A range from 1000 or greater reads of sequencing for short insert methods can be used for this method. Large insert methods such as Pac BioTm, NanoporenT, or other next gene sequencing methods can use <1000 sequencing reads. Bioinformatics quality filtering was performed before taxonomy assignment. Quality trimming of raw sequencing files may include removal of sequencing adaptors or indexes;
trimming 3' or 5' end of reads based on quality scores (Q20>), basepairs of end, or signal intensity;
removal of reads based on quality scores, GC content, or non-aligned basepairs; removal of overlapping reads at set number of base pairs. Alignment of processed sequencing files was done using a custom microbial genome database consisting of sequences from refseem, GreengeensTm, HMPTm, NCBITm, PATRICTm, or other public/private data repositories or in-house data sets. This database may be used as full genome alignment scaffold, k-mer fragment alignment, or other schemes practiced in the art of metagenomics and bioinformatics. Based off the number of sequencing reads/fragments that match the database genomes we assign a taxonomic identity that is common or unique to the organism. This identifier can be a barcode, nucleotide sequence, or some other computational tag that will associate the matching sequencing read to an organism or strain within a taxonomic group. Some identifiers will be of higher order and would identify domain, kingdom, phylum, class, order, family, or genus of the organism.

[0078] The present invention is able to identify the organism at the lowest order of strain within a species.

[0079] In embodiments 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, Bacillus amyloliquefaciens, Lactobacillus helveticus, Bacillus subtilis, Lactobacillus plantarum, Bifidobacterium longum subsp infantis, Enterococcus hirae, Lactobacillus delbrueckii subsp bulgaricus, Enterococcus, Lactobacillus rhamnosus, Lactococcus lactis, Pseudomonas stutzeri, Lactobacillus acidophilus, Klebsiella and Enterobacter cloacae strain.

[0080] In embodiments the invention includes identification and/or analysis of one or more yeast contained within our database (Figure 10). Some selected examples are Saccharomyces sp. Boulardii, Saccharomyces kudriavzevii, Saccharomyces pastorianus and Saccharomyces cerevisiae.

[0081] In embodiments the invention includes identification and/or analysis of one or more phage or viruses contained within our database (Figure 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.

[0082] Future database improvements will increase or refine the organisms that can be detected by this method.

[0083] In an embodiment, the extracted and purified genetic material was prepared for sequencing using Illumina index adaptors and checked for sizing and quantity.
Low cycle PCR may be performed or standard PCR-free methods. Gel purification was performed using the Qiagen Gel Purification KitTm (Qiagen, Frederick, MD). Clean PCR
products were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA).
Samples were combined in equimolar amounts. Library pools were size verified using the Fragment AnalyzerTm CE (Advanced Analytical Technologies Inc., Ames IA) and quantified using the Qubiti'm High Sensitivity dsDNA kit (Life Technologies, Carlsbad, CA). After dilution, a 10% spike of Phi)014 V3 library control (IIlumina, San Diego CA), pools were denatured for 5 minutes in an equal volume of 0.1 N NaOH then further diluted in Illumina's HT1 buffer. The denatured and PhiXTm-spiked pool was loaded on an Illuminarm Next Generation Sequencer with Illumina sequencing primers and set for 150 base, paired-end reads. Bioinformatics quality filtering was performed before taxonomy assignment.

[0084] Using Table 1, we determine that the individual has consumed the following:
Table 1 Taxon Reference Species reads reads subtree ablvidances abundances Level ID Name subtree rcestimated subtrce subtree reestimated 4513 Hordeum 684758 651478 4.56 77.33 vulgare 9858 C,apreolus 1522 1522 0.09 11.10 capreolus 3276 Rapitanus 179566 179566 10.21 36.03 salivus 161934 Beta 3251 0 0.17 11.21 vulgaris 4565 Tritictan 32243 32243 1.24 32.47 aestivum 93759 Col-chorus 23293 23293 0.15 18.56 olitorius 9796 Equus 69875 69875 0.43 11.49 cabailus 4120 Tponloe.a 5374 5374 0.13 11.71 baiatas MONITORING MACRONUTRIENT INTAKE AND DIETARY GUIDANCE

[0085] In some embodiments, the present invention may be used to monitor food intake nutrition, quantity, and quality in subjects. For example, prior to treatment with a probiotic, a sample obtained from the digestive tract of a subject may be obtained and the genetic material of the food organisms therein extracted as disclosed herein and subjected to metagenomics analysis. A customized food specific database comprised of whole, partial, or incomplete reference genomes, RNA's, or nucleic acid components or fragments will be utilized by bioinformatics tools to identify, quantify, and taxonomically assign the nucleic acid information from sequencing. The output of which is exemplified in Table 2 below and contains identification of the species of organisms or cells of organisms that were in the gut.
Table 2 In Grams As a percent of daily recommended Scientific Name Common Name Relative Abundance Fats Carbohydrates Proteins Iron Vitamin A Vitamin B Vitamin C Vitamin D
Capreolus capreolus Deer 005 27 0 26 21 0%
Raphanus satwus Radthsh 005 0 02 0 0 0% 10 0%
Beta vulgans Beet 005 02 13 2 2 6 0% 5 0%
11 0%
Trfficum aestwum Wheat 0 3 47 137 26 37 0% 40 0%
Corchoms Worms Jute 005 02 0 6 15 0% 90 0% 25 0%
47 0%
Bos Taurus Cattle 0 4 6 0 22 15 0%
Ipomoea batatas Sweet Potato 0 1 0 1 27 2 1 4 0% 377 0% 15 0% 5 0%

[0086] Then during and/or after treatment with a given probiotic, a second sample may be obtained from the digestive tract of the subject and the genetic material of the microbes in the second sample extracted and subjected to metagenomics analysis, the results of which are compared to the results of the metagenomics analysis of the first sample. Then, based on the comparative results, the food organism results maybe compared to the microbiome organism results to understand the microbes associated with food and an overall food quality assessment. In some embodiments, this may provide information to the species of organism that an individual is ingesting through their food source and any genetic modifications, mutations, or irregularities to the species either by selection or direct modification.

[0087] In some embodiments, the second sample of microbiome analysis will enable detection of microbes common to the food organisms and provide information on the health of the food organism. In some embodiments, the human consumed food may be part of the common food source such as chickens, cows, pig, or even plants, and protista where the species will be identified and match to microbes that are specific to them. In particular example embodiments, a chicken species that may have a chicken sarcoma virus may be detected in the second gut microbiome sample analyzed. In some embodiments, the health of a food organism ingested can be determined by the presence or absence of microbes that negatively impact the health of the host organism. In particular example embodiments, a disease, such as Equid herpesvirus 2, which is a respiratory disease in horses, may be detected that may impact the health of a host organism.

[0088] In some embodiments, the present invention may be used to screen the gut microbiome of a given subject and then custom tailor a food or diet regime that would enable them to improve the quality of their health for aspects of nutritional balance, improved microbial gut profile, and absorption of nutrients.
MONITORING PROBIOTIC TREATMENT

[0089] In some embodiments, the present invention may be used to monitor probiotic treatment in subjects. For example, prior to treatment with a probiotic, a sample obtained from the digestive tract of a subject may be obtained and the genetic material of the microbes therein extracted as disclosed herein and subjected to metagenomics analysis.
Then during and/or after treatment with a given probiotic, a second sample may be obtained from the digestive tract of the subject and the genetic material of the microbes in the second sample extracted as disclosed herein and subjected to metagenomics analysis, the results of which are compared to the results of the metagenomics analysis of the first sample. Then, based on the comparative results, the probiotic treatment of the subject may be modified to obtain a desired population of microbes in the gut of the subject. For example, a probiotic that comprises a microbe whose amount is desired to be increased in the gut of the subject may be administered to the subject.

[0090] In some embodiments, the fecal sample may be mixed or cultured for determination of metabolomic of microbial fecal community. Metabolomic profile can then be used to determine probiotic strains that would benefit the individual.
Examples of metabolomic profiles include those affecting energy metabolism, nutrient utilization, insulin resistance, adiposity, dyslipidemia, inflammation, short-chain fatty acids, organic acids, cytokines, neurotransmitters chemicals or phenotype and may include other metabolomic markers.
MICROBIOME SCREENING AND PROBIOTIC SELECTION

[0091] The present invention has been successfully used to determine the microbe content of a variety of commercially available probiotics. Additionally, the methods of the present invention are used to determine the microbe content of various probiotics and the microbiome content in the gut of the subject. In one embodiment, based on the microbiome content in the gut of the subject and any desired changes thereto, one may select one or more probiotics that contain the microbes that are desired to be increased and/or maintained in the subject's microbiome health. In one embodiment, based on the microbiome content in the gut of the subject and any desired changes thereto, one may select one or more probiotics that contain the microbes that are desired to be increased and/or maintained in the subject's gut balance in relation to the macronutrient content they are getting from their food source as recorded by survey information from the individual directly or by the present invention of gut organism nucleic acid analysis.

[0092] Where the microbiome represents a full picture of their microbiota and the organisms contained in them from 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 the subject's gut microbiome is desired to be maintained, one would select Probiotic 2 for administering to the subject.
However, if the amounts of A and B in the subject's gut are desired to be 50/50, one may select both Probiotics 1 and 2 to be administered to the subject.
Alternatively, one may select Probiotic 1 to be administered to the subject until the amounts of A
and B in the subject's gut reaches 50/50. In some embodiments, one may custom tailor a probiotic formulation, e.g., containing equal, varying, or diverse amounts of A and B or other probiotic strains, for administration to the subject. Calculation models utilizing relative abundance of the microbes present in an individual's gut will help determine the type, dose, and cocktail of microbes to include in the probotic. For example, if it is determined that organism A is reduced or absent compared to the general population or previous microbiome analysis, then we would provide probiotic or prebiotics that would increase the concentration of organism A. This prebiotic or probiotic may be the exact organism A
or another organism what would support the grown of organism A. The dose given would consider relative abundance of organisms in the individual, performance characteristics of the prebiotic/probiotic such as growth rate, compatibility, receptors or receptor density, genes, or expression patterns, or metabolomic products.

[0093] Custom tailored probiotics may not be in equal amounts but are formulated based on relative abundance detected from the individual gut/fecal sample.
These formulations are geared to modulate the microbiome to a healthy status. The healthy status of a microbiome is determined by the use of existing aggregate private and public databases such as metaHIT", Human Microbiome Project', American Gut Project", and the like. The healthy status may also be determined individually when a person has no known issues and is in good health, from a blood biomarker checkup perspective, and then has their full microbiome profile completed. After one or several microbiome signatures have been completed then the average of some/all of the microbes found can be understood for that individual and variances from that average can be accessed to determine if they are in dysbiosis. Microbiome profiles can be aggregated into groups that are then assigned a barcode for rapid bioinformatic assignment. Groups can be created by single or multiple phenotypic, diagnostic, or demographic information related to the individual from which the sample was collected from. A unique group can be determined from another group by using statistical models such as linear distance calculations, diversity values, classifiers such as C4.5 decision tree, or principal component analysis an comparing to an aggregate known population such as "normals" defined by the Human Microbiome Project or American Gut Project.

[0094] Thus, in some embodiments, the present invention may be used to screen the gut microbiome of a given subject and then custom tailor a probiotic regimen to the given subject based on the subject's gut microbiome.

TREATMENT OF DYSBIOSIS

[0095] In some embodiments, the present invention may be used to restore a subject's gut flora and/or fauna to homeostasis after an event that has caused a shift in the subject's microbiota from balanced microbiome to one that is causing or may be causing negative side effects, disorders, and/or disease. Health conditions can include but is not limited to various conditions, from acne and allergies, through gastrointestinal ailments, obesity and cancer. One example of such a dysbiosis is in the case of the onset of obesity. Several strains of microbes in the guts of subjects have been shown to be associated with obesity or weight management issues suffered by the subjects. See, e.g., Ley, et al.
(2005) PNAS
USA 102:11070-11075. For example, in obese animal and human subjects, the ratio of Bacterides to Firmicutes phyla microbes plays an important role in metabolic performance. See, e.g., Tumbaugh, et al. (2012) PLOS ONE 7:e41079. Some gut microbes known to be associated with obesity and weight management issues include Bacteroides uniformis, Bacteroides pectinophilus, Roseburia inulinivorans, Methanobrevibacter smithii, and Bifidobacterium animalis.

[0096] Thus, in some embodiments, a ratio of a first given microbe to a second given microbe in the gut of a subject is determined using the methods described herein and then if the ratio is undesired or abnormal, the subject is administered a treatment to modify the ratio to be a desired ratio. In some embodiments, the amount of a first given microbe in a gut of a subject relative to the total amount of all the microbes in the gut of the subject is determined using the methods described herein and then if the relative amount of the first given microbe is undesired or abnormal, the subject is administered a treatment to modify the amount to be a desired amount. Re-testing of their gut microbiome maybe used to determine well they are adhering to the macronutrient and food guidance. Such treatments include administering to the subject: a probiotic containing one or more microbes whose amounts are desired to be increased in the gut of the subject, an antimicrobial agent, e.g., an antibiotic, an antifungal, an antiviral, etc., to kill or slow the growth of a microbe or microbes whose amounts are desired to be decreased in the gut of the subject, a diet and/or a dietary supplement that supports the growth or maintenance of a healthy gut microbiome, e.g., a prebiotic, magnesium, fish oil, L-glutamine, vitamin D, etc., and the like. For example, Million, et al. ((2005) Int. J. Obes. 36:817-825) indicate that the gut microbiota of obese subjects are enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii. Therefore, after determining the amounts of Lactobacillus reuteri, Bifidobacterium animalis, and Methanobrevibacter smithii in the gut of a subject using the methods described herein and finding that the amounts are typical or indicative of obesity-associated gut microbiota, the subject may be administered a probiotic containing Bifidobacterium animalis and Methanobrevibacter smithii and relatively little to no amount of Lactobacillus reuteri. In embodiments, the gut microbiota of obese subjects would benefit from foods with flavonoids, polyphenols, and short chain fatty acids.
SCORING OF YOUR MICROBIOME

[0097] Scoring of the microbiome signature overall uses a similar decision tree, algorithm, artificial intelligence, script, or logic tree as represented in Table 3. This system would enable a score that helps a user understand how healthy their gut microbiome is and if they need to take action on a few or many challenges found.
Challenges can include but not limited to, identification of known pathogenic organisms, count and identification of opportunistic pathogens, latent organisms known to cause pathogenic affects when given opportunity, lack of support for good microbial environment but their composition or lack of key strains, overall diversity and count of unique organisms found in top 10 and or organisms with greater than 0.1%
prevalence.

[0098] Diversity cut offs were determined from an aggregate of sample analysis and a cutoff is determined at x relative abundance. For example, if x= 0.1% then 352 unique organisms make up the average healthy profile. Then apply standard deviations around this number and using a Gaussian distribution and percentile under the curve analysis we can score how close to the average diversity number from our database average.
The lower your diversity number and further away from the average you are then the less that microbiome would score. The higher the number and the greater your diversity is the more that microbiome would score. This type of scoring categories along with probiotic score will determine a number and visual metered score for the custom to understand how healthy their microbiome is. An example of the graphic visualization is included below.

Where low is equal to low microbiome quality and high is equal to high microbiome quality and score. Low - > 30 out of 100, Med > 65 out of 100, High = 65 or greater out of 100.

[0099] An example of a scoring and probiotic formula algorithm is included in Table 3 below. Table 3 can be represented as decision tree, algorithm, artificial intelligence, script, or logic tree. The function of such decision tree, algorithm, artificial intelligence, script, or logic tree would be output a score of wellness of the individual microbiome as related to probiotics detected and to provide formulation and dosing recommendations for probiotic usage.

[0100] An exemplary list of potential categories into which microbes may be grouped is set forth in Table 4 below.

Table 3 Example Decision Table for Probiotic Scoring and Formulation.
Includes the Utilization of a Probiotic Strain Database, Metagenomic Analysis Database, and Literature Curation Database Criteria Criteria Criteria Score or Inclusion/Exclusion Number Answer 1 Greater than 100 reads Yes If yes then include Greater than 50% of 2 Yes total probiotic reads Greater than 10,000 3 Yes If yes do not include in probiotic formula reads Greater than 50% of 4 No total reads Greater than 30,000 Yes If yes do not include in probiotic formula reads Greater than 30,000 6 reads for x number of Yes If x>5 then score +20, x>3 score 10, x>1 score 5 probiotics Total number of If x>10 then score +20, x>10 then score 10, x>5 7 microbes above 100 x score 5 reads (count) Query for probiotic strains and output where 1=yes and 4 is 8 Yes Include in formula at 20CFU/g or greater no and 6 is no and the number of reads is less than 1000 9 If bacillus Yes Do not include If lactobacillus If x> 10000 score +20, if x>1000 score +10, if x acidophilus greater Yes >100 score +5 than x reads If bacillus genus If x> 1000 score +20, if x>100 score +10, if x 11 Yes greater than x reads >10 score +5 If Saccharomyes If x> 1000 score +20, if x>100 score +10, if x 12 boulardi greater than x Yes >10 score +5 reads If infant if nursing and If x>10 then score +5, x>30% then score +10, 13 bifidobacterium Yes x>50% then score +20, x>70% then score +30 infantis >x%
If not infant, not child If x>20 then score +5, if x>10 then score +10, if 14 and bifidobacterium Yes x<10 then score +20 infantis >x%
Query to probiotic function, if function table is equal to health phenotype or healthDx then include in formula unless 3 or 5 = yes Table 4 Potential Categories from which to Create Groups t..) o ,-, ,-, Categoriesl Categories2 Categories3 Categories4 Categories5 Categories6 CA
AC ID_REFLUX FLOSSING_FREQUENCY SC IENTIFIC_NAME
VIOSCREEN_D_YOGURT VIOSCREEN_M_MEAT .. VIOSCREEN_VITB12 .. W
.6.
ACNE_MEDICATION FLU_VACC I NE_DATE SEAFOOD_FREQUENCY
VIOSCREEN_EER VIOSCREEN_M_MPF VIOSCREEN_VITB6 N
ACNE_MEDICATION_OTC FROZEN_DESSERT_FREQU SEASONAL_ALLERGIES VIOSCREEN_EMAIL
VIOSCREEN_M_NUTSD VIOSCREEN_VITC
ENCY
ADD_ADHD FRUIT_FREQUENCY SEQUENCING_METH
VIOSCREEN_ERYTHR VIOSCREEN_M_ORGAN VIOSCREEN_VITD
AGE_CAT FUNGAL_OVERGROWTH SEX
VIOSCREEN_FAT VIOSCREEN_M_POULT VIOSCREEN_VITD2 AGE_CORRECTED GEO_LOC_NAME shannon_10k VIOSCREEN_F_CITMLB
VIOSCREEN_M_SOY VIOSCREEN_VITD3 AGE_YEARS GLUTEN shannon_1k VIOSCREEN_FIBER
VIOSCREEN_MULTI_CALCIU VIOSCREEN_VITD_IU
M_AVG
ALCOHOL_CONSUMPTION HAS_PHYSICAL_SPECIMEN SIBO VIOSCREEN_FIBH20 VIOSCREEN_MULTI_CALCIU VIOSCREEN_VITE_IU
M_DOSE
ALCOHOL_FREQUENCY HEIGHT_CM SKIN_CONDITION VIOSCREEN_FIBINSO
VIOSCREEN_MULTIVITAMIN VIOSCREEN_VITK
ALCOHOL_TYPES HEIGHT_UNITS SLEEP_DURATION VIOSCREEN_FINISHED
VIOSCREEN_MULTIVITAMIN
VIOSCREEN_V_ORANGE P
_FREQ

L.
ALCOHOL_TYPES_BEERC ID HIGH FAT_RED_MEAT_FRE SMOKING_FREQUENCY
VIOSCREEN_FISH_SERVING VIOSCREEN_NATOCO VIOSCREEN_V_OTHER 0 ER QUENCY S
u, ..]
a.
ALCOHOL_TYPES_RED_WIN HOMECOOKED_MEALS_FRE SOFTENER VIOSCREEN_F_NJ_CITMLB
VIOSCREEN_NCCGLBR VIOSCREEN_V_POTATO u, E QUENCY
n, ---.1 ALCOHOL_TYPES_SOUR_B HOST_COMMON_NAME SPECIALIZED_DIET
VIOSCREEN_F_NLOTHER VIOSCREEN_NCCGLGR VIOSCREEN_V_STARCY n, EERS
o 1 ALCOHOL_TYPES_SPIRITSH HOST_SUBJECT_ID SPECIALIZED_DIET_EXCLU
VIOSCREEN_F_NJ_TOTAL VIOSCREEN_NIAC IN VIOSCREEN_V_TOMATO
L.
ARD_ALCOHOL DE_DAIRY

ALCOHOL_TYPES_UNSPECI HOST_TAXID SPECIALIZED DIET_EXCLU
VIOSCREEN_FOL_DEQV VIOSCREEN_NIACINEQ VIOSCREEN_V_TOTAL
FIED DE_NIGHTSHADES
ALCOHOL_TYPES_WHITE_ IBD SPECIALIZED DIET_EXCLU
VIOSCREEN_FOL_NAT VIOSCREEN_NITROGEN VIOSCREEN_WATER
WINE DE_REFINED_SUGARS
ALLERGIC_TO IBD DIAGNOSIS SPECIALIZED_DIET_FODMA VIOSCREEN_FOL_SYN
VIOSCREEN NON_FRIED_FI VIOSCREEN_VVEIGHT
P SH
SERVINGS
ALLERGIC TO I HAVE NO_ IBD_DIAGNOSIS_REFINED
SPECIALIZED_DIET_HALAAL VIOSCREEN_FORMONTN VIOSCREEN NUTRIENT_RE
VIOSCREEN_WGRAIN
FOOD ALLERGIES_THAT_I_ COMMENDATION
KNOW OF
ALLERGIC_TO_OTHER IBS SPECIALIZED DIET_I DO_N VIOSCREEN_F_OTHER
VIOSCREEN_OMEGA3 VIOSCREEN WHOLE_GRAI IV
OT_EAT_A_SPECIALIZED_DI
N SERVINGS n ET
ALLERGIC_TO_PEANUTS INSTRUMENT_MODEL SPECIALIZED_DIET_KOSHE
VIOSCREEN_FRIED_FISH_S VIOSCREEN_OXALIC VIOSCREEN_XYLITOL
CP
R ERVINGS
N

Claims (35)

What is claimed is:

1. A method, comprising:
a) mixing a sample with a liquid nitrogen solution;
b) adding a first lysis solution, the first lysis solution comprising a detergent and a chelator; and c) adding a second lysis solution, the second lysis solution comprising a chaotropic agent.

2. The method of claim 1, wherein the mixing the sample with a liquid nitrogen solution further comprises grinding the sample and the liquid nitrogen mixture.

3. The method of claim 1, wherein the first lysis solution further comprises one or more buffers, one or more mild detergents, and/or one or more proteases.

4. The method of claim 1, wherein the detergent of the first lysis solution comprises SDS.

5. The method of claim 4, wherein the SDS concentration is about 10%.

6. The method of claims 1, wherein the chaotropic agent comprises lithium acetate.

7. The method of claim 6, wherein the mixture of sample, liquid nitrogen, the first lysis solution and the second lysis solution is further subjected to a heat shock treatment.

8. The method of claim 1, wherein the sample is subjected to a pretreatment step before treatment with the first lysis solution, said pretreatment step induces germination of any bacterial spores and/or fungal spores present in the sample.

9. The method of claim 8, wherein the pretreatment step comprises mixing the sample with Tween-80.

10. The method of claim 1, further comprising a mechanical treatment step that causes physical lysis, said mechanical treatment step comprises sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, or combinations thereof

11. The method of claim 1, further comprising subjecting any extracted genetic material metagenomics analysis.

12. The method of claim 1, wherein the sample is obtained from the gut of a subject.

13. The method of claim 11, wherein the metagenomics analysis identifies one or more foods that the subject has consumed.

14. The method of claim 11, wherein the metagenomics analysis identifies one or more macronutrients that the subject has consumed.

15. The method of claim 11, further comprising determining a probiotic treatment for the subject based on the metagenomics analysis.

16. The method of claim 11, further comprising determining a dietary guidance for the subject based on the metagenomics analysis.

17. A method of determining food consumption of a subject comprising:
a) extracting genetic material from a stool sample obtained from the subject, said genetic material extracted according to claim 1; and b) subjecting the genetic material extracted from the first sample to metagenomics analysis to determine the food consumption of the subject.

18. The method of claim 17, further comprising treating the subject with a probiotic or a food stuff based on the analysis of food consumption.

19. The method of claim 17, wherein metagenomic analysis comprises use of a database having genomic data of organisms useful for identification of such organisms.

20. The method of claim 19, wherein the database may be processed as whole genomes, k-mers of various lengths that are common to a higher order and unique to a specific one, or other means of barcoding genomes to match them to sequencing results.

21. The method of claim 17, wherein metagenomic analysis comprises preprocessing of sequencing information selected from removing duplicates, removing adaptor sequencing, removing 5' or 3' sequencing to improve the quality of base calling, including only base calls of a particular quality (i.e., Q20 or greater), filtering human reads, creating paired reads or separating them, and limiting overlap of reads.

22. The method of claim 19, wherein metagenomic analysis comprises aligning sequencing information to the database by use of a software or system where the sequencing information may be broken into k-mers of particular length, used as full fragments, be scaffolded and aligned to a large reference genome, or other method to create a report of organisms identified, relative abundance of organism identified, genome size, total fragments aligned, unique fragments aligned at the strain, species, genus, family, order, class, phylum, kingdom, or domain.

23. The method of claim 17, wherein microbiome profiles enable identification of a disease, disorder, or specific signature indicating a dysbiosis where probiotic and/or dietary supplement treatment can be applied to modulate the microbiome to improve the underlying profile.

24. The method of claim 23, wherein a group is created based on demographic, phenotypic, or diagnostic information and a barcode assigned to that group of profiles.

25. The method of claim 24, wherein statistical analysis is used to determine how closely related the individual microbiome profile is to an known group in a database.

26. A method of monitoring a probiotic treatment of a subject which comprises:
a) extracting genetic material from any microbes present in a first sample obtained from the subject, said genetic material extracted according to claim 1;
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 microbes present in a second sample obtained from the subject in the same manner as the extraction of genetic material from the first sample;
d) performing metagenomics analysis on the extracted genetic material from the second sample; and e) comparing the results of the metagenomics analysis of the first sample with the metagenomics analysis of the second sample.

27. The method of claim 26, which further comprises modifying the probiotic treatment to obtain a desired microbe population within the subject.

28. The method of claim 26, further comprising analysis of metabolomic markers to determine appropriate probiotic treatment.

29. The method of claim 26, wherein probiotic treatment follows use of an antibiotic, chemotherapy, a pharmaceutical, environmental change, traveling, contaminate digestions, infarction of the intestines or gut, stress, or other effect which disruption of the microbial population can occur.

30. The method of claim 27, wherein modulation of the microbiome is to return an individual to a previous population of microbes of when the individual was known to be healthy.

31. The method of claim 27, wherein modulation of the microbiome by a probiotic and/or a prebiotic is to restore microbiome profile to a normal gut that has been defined either internally by a database or externally by a public database.

32. The method of claim 31, wherein normal is defined as similar to the microbiome profile of a fecal matter repository sample used in fecal matter transplants, but used as a probiotic/prebiotic to restore a dysbiosis.

33. A computing system comprising: a memory; and one or more processors coupled to the memory, the one or more processors configured to perform operations to perform the method of claim 17.

34. A computing system comprising: a memory; and one or more processors coupled to the memory, the one or more processors configured to perform operations to perform the method of claim 26.

35. An automated platform for performing the method of claim 1.