CA3149221A1 - Method of testing for specific organisms in an individual - Google Patents

Abstract

A method of testing for specific organisms in an individual comprising the steps of: a) screening the individual; b) acquiring a stool sample from the individual; c) processing the stool sample to obtain the individual's microbiome; d) sequencing the microbiome of the individual; and e) analyzing the microbiome of the individual to determine whether one or more specific organisms are present in the individual, whereby a health condition of the individual is determined. The step of processing can comprise the sub-steps of: i) extracting DNA from the stool sample, which comprises adding the stool sample to a bead beating tube, achieving cell lysis, capturing the DNA on a silica membrane in a spin-column, and washing and eluting the DNA from the membrane; and ii) purifying the extracted DNA. A method of determining whether an individual has a health condition comprising the same steps. A stool sample collection kit.

Description

METHOD OF TESTING FOR SPECIFIC
ORGANISMS IN AN INDIVIDUAL

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 METHOD OF TESTING FOR SPECIFIC ORGANISMS IN AN INDIVIDUAL
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001) This application claims priority to United States Provisional Patent Application No. 62/882,367, titled "Method of Analyzing the Microbiome of Individual Stool Samples,"
filed August 2, 2019, United States Provisional Patent Application No.
62/892,409, titled "Method of Analyzing the Microbiome of Individual Stool Samples," filed August 27, 2019, United States Provisional Patent Application No. 62/991,190, titled "Method of Analyzing the Microbiome of Individual Stool Samples," filed March 18, 2020, and United States Provisional Patent Application No. 63/002,486, titled "Method of Analyzing the Microbiome of Individual Stool Samples," filed March 31, 2020. The contents of all the applications are incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[00021 The human gastrointestinal (GI) microbiome is a complex, interconnected web of microbes, living in a symbiotic relationship with their host. There are greater than ten times more bacteria in our bodies than there are human cells, all in a delicate and ever-changing balance to maintain a healthy GI tract. When this balance is disrupted, a condition known as dysbiosis, or disease, can occur. There is still a debate over whether dysbiosis is a cause of disease or a symptom of it. Natural'), since the microbiome has such a profound impact on human health, including helping us digest food, make vitamins, and teach our immune cells to recognize pathogens, there is a desire study and learn as much about the microbiome as possible.
[0003] By correlating the microbiome data with survey data and medical records for the patients, connections may begin to be drawn between organisms present in the microbiome of the gastrointestinal tract, and a corresponding disease. For example, if there is one particular PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 microbe in patients with Crohn's disease, the data suggest that this microbe could play a role in the cause or progression of this disease.
[0004] Accordingly, there is a need for a method of testing for specific organisms so that appropriate treatment may be rendered. The present invention satisfies this need.
SUMMARY
(0005) In a first embodiment, the present invention is directed to my method of testing for specific organisms in an individual. The method comprises the steps on a) screening the individual; b) acquiring a stool sample from the individual; c) processing the stool sample to obtain the individual's microbiome; d) sequencing the microbiome of the individual; and e) analyzing the microbiome of the individual to determine whether one or more specific organisms are present in the individual, whereby a health condition of the individual is determined.
100061 The step of processing can comprise the sub-steps of i) extracting DNA from the stool sample, which comprises adding the stool sample to a bead beating tube, achieving cell lysis, capturing the DNA on a silica membrane in a spin-column, and washing and eluting the DNA from the membrane; and ii) purifying the extracted DNA.
[0007] Optionally, step b) comprises providing the individual with a stool sample collection kit.
[0008] The stool sample collection kit can comprise a) at least one stool sample collection vial; b) at least one toilet accessory, c) at least one specimen bag; d) at least one pair olgloves: e) an authorization form; I) a patient information card; g) a questionnaire; and h) stool sample collection instructions.
[0009] Optionally, step b) comprises acquiring the stool sample from the individual via colonoscopy

2 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 2042553 (00101 The one or more specific organisms o f step e) can comprise one or more of the following: Acinetobacter baumannii, Actinomyces odobtolyticus, Akkermansia muciniphila, Bacillus cereus, Bacillus subtilis, Bactetiodes fragilis, Bacteroides vulgatus, Bifidobacterium adolescent, Blastocystis hominis, Butyrivibrio proteoclasticus, Campylobacter jejuni, Candida albicans, Chlamydophila pneumoniae, Clostridioides difficile, Clostridium beijerinckii, Clostridium perfiingens, Clostridium sporgesse, Crptococcus neoforimans, Cutibacterium acnes, Deinococcus radiodurans, Enterobacter cloacae, Enterococcus faecalis, Eseherichia coli, Fusobacterium nucleatum, Helicobacter hepaticus, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus gassed, Lactobacillus fermenturn, Lactobacillus plantarum, Listeria rnonocytogenes, Mycobacterium aviurn subsp.
paratuberculosis, Neisseria 'meningitides, Porphyromonas gingivalis, Proteus mirabilis, Pseudomonas aeruginosa, Rhodobacter sphaeroides, Saccharomyces cerevisiae, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus mutano, Streptococcus pneumoniae, Streptococcus pyog,enes, Toxoplasma goridii, Versinia enterocolitica, and Bacteria X.
100111 Optionally, step e) is an assay that tests for the following organisms:
Acinetobacier baumannii, Actinomyces odontolyticus, Akkermansia muciniphila, Bacillus cereus, Bacillus subtilis, Bacteriodes fragilis, Bacteroides vulgatus, Bifidobacterium adolescent, Blastocystis hominis, Butyrivibrio protooclasticus, Campylobacter jejani, Cartdida albicans, Chlamydophila pneurnoniae, Clostridioides difficile, Clostridium beijerinckii, Clostridium perfringens, Clostridium sporgesse, Crptococcus neoformans, Cutibacterium acnes, Deinococcus racliodurans, Enterobacter cloacae.
Enterococcus faecalis, Eschenchia coil, Fusobactenurn nucleatum, Helicobacter hepaticus. Helicobacter pylori, Klebsiella pneumoniae. Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus plantarum, Listeria monocytogenes, Mycobacterium avium subsp.
paratuberculosis, Neisseria

3 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No, 20-42553 meningitides, Porphyromonas gingivalis. Proteus mirabilis, Pseudomonas aeruginosa, Ilhodobacter sphaeroides, Saccharomyces cerevisiae, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus miXano, Streptococcus pneurnoniae, Streptococcus pyogenes, Toxoplasina gondii, Yersinia enterocolitica, and Bacteria X.
100121 Optionally, step e) comprises comparing the microbiome of the individual to a microbiome of a mother of the individual.
100131 Optionally, step e) comprises comparing the microbioine of the individual to a microbiome of a sibling of the individual.
100141 Optionally, step e) comprises comparing the tnicrobiotne of the individual with a health condition to a microbiome of another individual with the same health condition.
100151 Optionally, step e) comprises comparing the microbiome of the individual with a health condition to a microbiome of the individual before the individual had the health condition.
(00161 The method can further comprise step 0 after step e), storing the processed stool sample in a freezer.
[00171 In a second embodiment, the present invention is directed to a method of determining whether an individual has a health condition. The method comprises the steps of a) acquiring a stool sample from the individual; b) processing the stool sample to obtain the microbiome; c) sequencing the microbiome of the individual; and d) analyzing the microbiome of the individual to determine whether one or more specific organisms are present in the individual, whereby the health condition of the individual is determined.
[00181 The health condition is selected from the group comprising. C. difficile infection.
Obesity, Autism, Alzheimer's disease, Crolm's disease, Myalgic Encephalomyelitis/Chronic,

4 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 Fatigue Syndrome (ME/CFS), Psoriasis, Chronic Urinary tract infection, Ulcerative Colitis, Multiple Sclerosis, Chronic constipation, Celiac sprue. Lyme disease, Elevated cholesterol, Colorectal cancer, Arnyotrophic lateral sclerosis, Rheumatoid arthritis, Parkinson's disease, Depression, Anxiety, Obsessive-Compulsive disorder, Bipolar Disorder, Migraine headaches, Diabetes mellitus, Lupus, Epidermolysis, Metastatic mesotheliotna, irritable bowel syndrome Diarrhea, irritable bowel syndrome Constipation, Eczema, Acne, Fatty liver, Myasthenia gravis, and Gout.
100191 Step b) can comprise the steps of:
[0020) i) extracting DNA from the sample, which comprises the steps of adding the stool sample to a bead beating tube, achieving cell lysis, capturing DNA on a silica membrane in a spin-column, and washing and eluting the captured DNA from the membrane:
and 10021.) ii) purifying the extracted DNA
[0022] The one or more specific organisms of step d) can be selected from the group consisting of: Acinetobacter baumannii, Act inomyces odontolyticus, Akkermansia muciniphila, Bacillus cereus, Bacillus subtilis, Bactenodes fragilis.
Bacteroids vulgatus, Bilidobacterium adolescent, Blastocystis ho minis, Butyrivibrio proteoclasticus, Campylobacter jejuni, Candida albicans, Chlamydophila pneumonia;
Clostridioides difTicile, Clostridium beijerinckii, Clostridium perfringens, Clostridium sporgesse, Crptococeus neoformans, Cutibacterium acnes, Deinocoecus radiodurans, Enterobacter cloacae, Enterococcus faecalis, Eschericliia coli, Fusobacterium nucleatum, lielicobacter hepaticus, lielicobacter pylori, Klebsiella pneurnoniae, Lactobacillus gasseri, Lactobacillus fermenturn, Lactobacillus plantarum, Listeria monocytogenes, Mycobacterium avium subsp paratuberculosis, Neisseria meningitides, Porphyromonas gingivalis, Protects mirabilis, PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 Pseudomonas aeruginosa, Rhodobacter sphaeroides, Saccharomyces cerevisiae.
Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus =tam, Streptococcus pneuinoniae, Streptococcus pyogenes, Toxoplasma gondii, Yersima enterocolitica, and Bacteria X.
DRAWINGS
10023.1 These and other features, aspects and advantages of the present invention will be better understood with reference to the following description, appended claims, and accompanying drawings where:
100241 FIG. I is a flow chart of a method of testing an individual for specific organisms having features of the present invention;
[0025] FIG, 2 is a top plan view of a stool collection kit having features of the present invention;
[00261 FIG. 3 is top plan view of the stool collection kit of FIG
2, wherein the contents have been removed from the box;
[0027] FIG. 4 is a graphical representation of the number of various mycobacterium found in the samples:
[0028] FIG. 5 is a graphical representation of the biodiversity of mycobacterium in healthy patients versus patients with Crohn's Disease of Example I ;
[0029] FIG. 6 is a graphical representation of the mycobacterium of patient 12 compared to patient 12's biological mother (patient 11) of Example 1:
100301 FIG. 7 is a graphical representation of mycobacterium of patient 2 compared to patient 2's biological mother (patient 1) of Example 1:
10031] FIG. 8 is a graphical representation of the mycobacterium of patient 10 versus patient 10's biological mother (patient 9) of Example 1:

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [0032] FIG. 9 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother (patient 11) of Example 1;
[0033) FIG. 10 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother (patient 11) of Example 1;
[0034] FIG. 11 is a graphical representation of a comparison of the microbiome between patient 2 and patient 2's biological mother (patient 1) of Example 1;
[0035] FIG. 12 is a graphical representation or a comparison of the microbiome between patient 2 and patient 2's biological mother (patient 1) of Example 1;
100361 FIG. 13 is a graphical representation of a comparison of the microbiome between patient 14 and patient 14's biological brother (patient 6) of Example 1:
100371 FIG. 14 is a graphical representation of a comparison of the microbiome between patient 10 and patient 10's biological mother (patient 9) of Example 1;
10038) MG. 15 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1, 10039] FIG. 16 is a graphical representation showing common organisms found in patients. with Crohn's disease of Example I;
100401 FIG. 17 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
100411 FIG. 18 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
[0042] FIG. 19 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
100431 FIG. 20 is a graphical representation showing common organisms found in patients with Crohn's disease of Example I;

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 100441 FIG. 21 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
[0045] FIG. 22 is a graphical representation of a comparison of the rnicrobiome between patient 1 and patient Vs biological mother of Example 1;
[0046] FIG, 23 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother (patient 11) of Example 1;
[0047] FIG. 24 is a graphical representation of a comparison of the microbiome between patient 2 and patient 2's biological mother of Example 1;
100481 FIG. 25 is a graphical representation ()fa comparison of the microbiome between patient 14 and patient 14's biological brother of Example 1:
[0049] FIG. 26 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother or Example 1;
[0050] FIG. 27 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1, [00511 FIG. 28 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
[0052] FIG. 29 is a graphical representation showing common organisms found in patients with Crohn's disease of Example 1;
100531 FIG. 30 is a flow chart of a method of testing an individual that was infected with COVID-19 of Example 10-, and I0054J fIG. 31 ia FIGS, 31A-31H are a series of graphs depicting whole genome alignment of SARS-CoV-2 in patients of Example 12.

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42533 DETAILED DESCRIPTION OF THE INVENTION
100551 The following discussion describes in detail one embodiment of the invention and several variations of that embodiment. This discussion should not be construed, however, as limiting the invention to those particular embodiments. Practitioners skilled in the art will recognize numerous other embodiments as well.
Definitions [00561 As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is clearly intended by the context in which such term is used.
100571 The terms "a," "an," and -the" and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise.
[0058] As used in this disclosure, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers, ingredients or steps.
THE INVENTION
100591 Referring now to Figure 1, the present invention is a method of testing an individual for specific organisms. The method comprises five main steps:
screening 100 the individual, acquiring 102 a stool sample from the individual. processing 104 the stool sample to obtain the individual's microbiome, sequencing 106 the microbiome of the individual, and analyzing 108 the microbiome of the individual to determine whether specific organisms are present in the individual.
[00601 During the step of screening, the individual typically undergoes the following:
signing of the consent form, providing their medical history and demographics, having their PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 vital signs taken/read. providing their height and weight, and providing the staff with a list of their prior and concomitant medications. Concomitant medications include any form of antibiotics, probiotics, or opiates.
10061] The individual then has a consultation to discuss the sequencing of their DNA
and the method used to collect the fecal sample. For individual who collect their stool samples at home, they are provided with a stool collection kit 200 (shown in Figures 2 and 3) and instructed in their use. Individual who will have their stool sample collected via colonoscopy provided with colonoscopy preparation instructions and a prescription for bowel cleanse. As standard-of-care, a gastroenterologist will collect the colonoscopy samples during a medically necessary colonoscopy.
[0062] The individual then completes demographic and medical history forms to generate data to accompany their microbiome sequencing data.
[0063] As noted above, the step of acquiring a stool sample can either involve the stool sample collection kit 200 or a colonoscopy The stool sample collection kit 200 is shown in Figures 2 and 3 and comprises: at least one stool sample collection vial 202, optionally the vial 200 contains a spoon, at least one toilet accessory or seal cover 204, at least one specimen bag 206, at least one pair of gloves 208, an authorization form 210, a patient information card 212, a questionnaire 214, and stool sample collection instructions 216.
10064j The toilet accessory 204 is in the form of a circular strip of paper that slips over the toilet seat and creates a raised platform on which to provide the voided stool sample.
100651 The stool sample collection instructions 216 are as follows: (I) Correctly position the toilet accessory (i.e. toilet cover) over the toilet seat and put on disposable latex gloves.
(2) Unscrew the collection tube cap and use the spoon to scoop one spoonful of the stool sample from the feces. Do not pass the stool sample into the toilet or directly into the collection vial, and do not mix urine or water with the stool sample. (3) Place the stool PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 sample into the collection vial. (4) Tighten the cap and shake to mix the contents thoroughly (and/or invert 10 times) to create a suspension. Some fecal material may be difficult to re-suspend. As long as the stool sample is suspended, the sample is stabilized.
Foaming/frothing during shaking is normal. (5) Place the collection vial in the bag labeled "Specimen Bag-Biohazard" and seal the bag. (6) Place the bag back in the collection kit box.
(7) Remove toilet cover and let it fall into the toilet bowl. Flush both the toilet cover and excess stool down the toilet. (8) Remove and dispose of gloves. Thoroughly wash hands.
I00661 Following collection of the stool sample, the stool sample is then processed and the microbiome analyzed. For these two steps, the following equipment is utilized:
centrifuges, pipettes, thermocycler, fluorometers, vortexers, refrigerators/freezers, and a sequencing system (for example, an Illumina NextSeq 550 Sequencing System) 100671 The step of processing the sample includes extracting and purifying patient DNA
from the sample. Individual patient DNA is extracted and purified with a DNA
extraction kit.
Optionally, the QIAmp`' PowerFecal' Pro DNA Kit can be used. The DNA
extraction kit isolates both microbial and host genomic DNA from stool and gut samples.
[0068] In summary, for the DNA extraction step, the stool samples are added to a bead heating tube for rapid and thorough homogenization. Cell lysis occurs by mechanical and chemical methods. Total genomic DNA is captured on a silica membrane in a spin-column format. DNA is then washed and eluted from the membrane and ready for NCiS, PCR and other downstream application [0069] Once the DNA has been extracted, the DNA is then quantitated using a fluorometer. The fluorometer can be a dual-channel fluorometer for nucleic acid quantitation.
It provides highly sensitive fluorescent detection Vv hen quantifying nucleic acids and proteins.
[00701 The following steps are performed when quantitating the sample:

PCT/us20/44605 26 May 2021 (26.05.2021) PCI APPLICATION
Attorney Docket No. 20-42553 [0071] Mix 1-20 microliters of the extracted DNA sample and 200 microliters of dye in a 0.5m! PCR tube. Mix well by pipetting or vottexing.
[0072] The fluorescence is then measured and the nucleic acid concentration is calculated and/or displayed.
[0073] Next, the library is prepared. The assay of the present invention is designed to detect all bacteria, viruses, and fungi that reside in the microbiome of the stool samples being evaluated. The assay utilizes an enzymatic reaction to fragment the DNA and to add adapter sequences. Library fabrication includes tagmentation, tagmentation clean-up, and an amplification step followed by another clean-up prior to pooling and sequencing.
100741 The following definitions and abbreviations are used in this section:
BLT: Bead-Linked Transposomes DNA: Deoxyribonucleic Acid EPM: Enhanced PCR Mix Et0H: Ethanol NGS: Next Generation Sequencing NTC: No Template Control PCR: Polymerase Chain Reaction RSB: Resuspension Butler SPB: Sample Purification Beads *FBI: Tagmentation Buffer TSB. Tagment Stop Buffer TWB. Tagrnent Wash Buffer 100751 First, the BLI and TBI are brought up to room temperature.
Then, the BLI and TB1 are vortexed to mix.

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [0076] Next, the appropriate volume of DNA is added to each well so that the total input amount is 100 nanagrams. The optimal input for this assay is 100 nanograins, however, less DNA input can be utilized.
100771 Next, the appropriate volume of nuclease-free water is added to the DNA samples to bring the total volume to 30 microliters.
100781 Then, the BLT is vortexed vigorously for 10 seconds. Next, 11 microliters of BLT
and 11 microliters of TB I are combined for each sample, creating a tagmentation rnastermix.
Overage is included in this volume.
100791 Next, the tagmentation master mix is vortexed and the volume is equally divided into an 8-tube strip.
100801 Next, 20 microliters of the tagmetnation master mix is transferred to each well containing a sample.
100811 Then, the plate is sealed with Microseal and placed on a thermo cycler preprogrammed with the TAG program The thermo cycler has a heated lid at 1000C
and reaction volume set to 50 microliters.
100821 Next, the TAG program is run as shown in Table 1:
Cyere. Step ¨77 Ieipperittit*',- 'T1.6"Oe =
Step 1 55 C 15 tninutes Step 2 10 C
Table 1 100831 Once the TAO program is complete, the plate is removed from the thermo cycler.
100841 Next, the Microseal 'B' seal is removed and 10 microliters ofTSB is added to each sample.
100851 Next, the plate is sealed with a Microseal 'B' and placed on the thermo cycler preprogrammed with the PTC program The therm cycler has a heated lid at I 00C.
10086] Next, the PTC program is shown in Table 2:

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 gt* 111*
Step I 37 C' 15 minutes Step 2 10T rkt Table 2 [0087] When the PTC program is complete, the plate is removed from the thermo cycler and placed on a magnetic stand. The plate is len on the magnetic stand for about 3 minutes (as long as it takes for the solution to clear).
[0088] Once the solution is clear, the Microseal 'B' is removed from the plate and the supernatant is removed and discarded.
[0089] Next, the plate is removed from the magnetic stand and about 100 microliters of TWB is added. The sample should be pipetted slowly until the beads are fully re-suspended.
[0090] Next, the plate is placed hack on the magnetic stand and approximately 3 more minutes pass while the solution clears again.
[00911 Once the solution clears, the supernatant is removed and discarded.
10092] Next, the plate is removed from the magnetic stand and about 100 microliters of TWII is added. The sample should be pipetted slowly until the beads are fully re-suspended.
10093] Next, the plate is again placed on the magnetic stand for an additional 3 minutes while the solution clears.
[0094] Next, 22 microliters of EPM and 22 microliters of nuclease-free water are combined with each sample to form a PCR mastermix. Overage is included in this volume. The PCR mastermix is vortexed and centrifuged [0095) With the plate on the magnetic stand, the supernatant is removed and discarded.
10096] Next, the plate is removed from the magnetic stand and 40 microliters of PCR
inasterrnix are immediately added directly onto the beads in each sample well [0097] The mastermix is immediately pipetted until the beads are fully re-suspended.
Alternatively, the plate is sealed and a plate shaker is used at 1600 rpm for I minute PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 100981 Next, the plate is sealed with a Microseal `.13' and centrifuged at 280 x g for 3 seconds.
100991 Next, 10 microliters of index adapters are added to each sample in the plate. The plate is then centrifuged at 280 x g for 30 seconds.
1001001 Next, the plate is placed on the thermo cycler that is preprogrammed with the BLT
PCR program (and with lid preheated at 100 C).
1001011 The BLT PCR Program is run as shown in Table 3:
= :Irikiiptr*4:::.õ-;4" ;,- !:µ:

-Slop 1 1 68 C 3 minutes Step 2 1 98 C 3 minutes = 98'C 45 seconds Step 3 5 62 C 30 seconds 68 C 2 minutes tI
Step 4 1 68 C 1 minute Step 5 I 10 C
Table 3 1001021 When BLT PCR program is complete, the plate is reinoµed from the therm cycler and centrifuged at 280 x g for I minute.
1001031 Next, the plate is placed on the magnetic stand and it takes about 5 minutes for the solution to clear.
1001041 Next, about 43 microliters of supernatant are transferred from each µvell ofthe PCR
plate to the corresponding well of a new midi plate.
100105] Then, the midi plate is vortexed and the SPB is inverted multiple times to re-suspend.
[00106] Next, about 40 microliters of nuclease-free water is added to each sample Well containing supernatant.

PC171.1520/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 1001071 Next, about 45 microliters of SPB is added to each sample well. Each sample well is then mixed.
[001081 The plate is then sealed and incubated for 5 minutes at room temperature.
1001091 Next, the plate is placed on the magnetic stand and it takes about 5 minutes for the solution to clear.
1001101 Next, the SPB is vonexed thoroughly and 15 microliters of SPB is added to each well ()la new midi plate.
1001111 Then, 125 microliters of supernatant is transferred from each well of the first plate into the corresponding well of the second midi plate containing 15 microliters SPB.
1001121 Each well of the second midi plate is then mixed and the first midi plate can be discarded.
[00113] The second midi plate is sealed and incubated for 5 minutes at room temperature.
[00114] The second midi plate is placed on the magnetic stand and it takes about 5 minutes for the solution to clear 100115] Next, µµ ithout disturbing the beads, the supernatant is removed and discarded.
100116j While the midi plate is still on the magnetic stand. 200 microliters of fresh 80%
Et0H are added to the plate, without mixing. The plate is then incubated for 30 seconds.
[001171 Next, without disturbing the beads, the supernatant is removed and discarded.
1001181 While the second midi plate is still on the magnetic stand, about 200 microliters of' fresh 80% Et0H are added, without mixing. The plate is then incubated for 30 seconds.
100119] Next, without disturbing the beads. the supernatant is removed and discarded. Any residual &OH is also removed and the second midi plate is allowed to air dry on the magnetic stand for about 5 minutes [001201 The second midi plate is remo ed from the magnetic stand and about 32 microliters of RSB is added to the beads.

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [001211 The second midi plate is then re-suspended and incubated for about 2 minutes at room temperature.
[001221 The second midi plate is placed back on the magnetic stand it takes about 2 minutes for the solution to clear.
[001231 Once the solution dears, about 30 microliters of supernatant are transferred to a new 96-well PCR plate.
1001241 Next, the library is pooled and sequenced.
1001251 The following definitions and abbreviations are used in this section:
DNA: Deoxyribonucleic Acid DOH: Ethanol HT1: Hybridization Buffer NGS: Next Generation Sequencing NTC: No Template Control RSB: Resuspension Buffer SAV: Sequencing Analysis Viewer 1001261 The following steps are taken to sequence the DNA:
1001271 Prepare the reagent cartridge for use.
1001281 Denature and dilute sample libraries.
1001291 Load pooled sample DNA libraries into the prepared reagent cartridge.
1001301 Set up and start the DNA sequencing using the selected DNA
sequencing machine, The sequencing run can take approximately 27-30 hours to complete.

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [001311 The bioinformatics pipeline utilizes a computational tool that profiles the microbial communities from metagenomic sequencing data with species level resolution.
Patient microbiome profiles are anab,zed to ascertain not only the profile of microbes in patient samples but also to identify specific strains, and provide accurate estimation of' organismal abundance relative to the overall diversity [001321 Once the DNA is sequenced, the microbiome the individual patient is screened using the assay of the present invention, as noted above. The assay tests for the Following organisms:
[00133] 1. Acinetobacter baumannii 100134] 2. Actinomyces odontolyticus [00135] 3. Akkermansia rnuciniphila [00136] 4. Bacillus cereus [00137] 5. Bacillus subtilis 1001381 6 Bacteriodes liagilis [00139] 7. Bacteroides vulgatus [00140] 8. Bifidobacterium adolescentis [00141] 9. Blastocystis hominis **(parasite) [00142] 10.Butyrivibrio proteoclasticus [00143] 11.Campylobacter jejtmi [00144] 12 Candida albicans [00145] 13. Chlarnyclophila pneumoniae [00146] 14.Clostridioides difficile 00147] 15. C. lostncliti m beijerinckii [00148] 16. Clostridium perfringens [00149] 17:Clostridium sporgesse PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [00150] 18.Crptococcus neoformans *(fungi) [00151] 19.Cutibacterium a,cnes [00152] 20.Deinococcus radio durans [00153] 21.Enterabacter cloacae [00154] 22.Enterococcus faecalis [00155] 23.Escherichia coli [00156] 24.Fusobacteriurn nucleaturn [00157] 25.14elicobacter hepaticus [00158] 26.1-1elicobacter pylori [00159] 27.Klebsiella pneumoniae [00160] 28.Lactobacill LIS gasseri [00161] 29.Lactobacillus ferment=
[00162] 30,Lactobacillos plantarum 1001631 3I,Listcria monocytogencs [00164] 32.Myeobacterium avium subsp. paratuberculosis [00165] 33,Neisseria meningitidis I00166J 341.Porphyromonas gingivalis [001671 35.Proteus mirabilis [001681 36.I'seudomonas aeruginosa [00169] 37.Rhodobacter sphaeroides 100170] 38.Saccharomyces cerevisiae *(fungi) [001711 39.Salmonella enterica 100172] 40,Staphylococcus aureus [00173] 4 I.Staphylococcus epidermidis 1001741 42.Streptococcus agalactiae PCT/us20/44605 26 May 2021 (26.05.2021) ACT APPLICATION
Attorney Docket No. 20-42553 [001751 43.Streptococcus mutano 1001761 44. Streptococcus pneumoniae 1001771 45.Strepto coccus pyogenes 1001781 46.Toxoplasma gondii **(parasite) 1001791 47.Yersinia enterocolitica 1001801 48. Bacteria X
1001811 The step of analyzing the microbiome of the individual can include the following: comparing the microbiome of the individual to the microbiome of the individual's mother, comparing the microbiome of the individual to the microbiome of a sibling of the individual, comparing the microbiome of the individual with a health condition to the microbiome of another individual with same health condition, and comparing the microbiome of the individual with a health condition to the microbiome of the individual before they acquired the health condition (otherwise referred to as baseline versus non-baseline).
[00182] lithe individual's baseline microbiome is being used m the analysis step, then the above recited steps of acquiring a stool sample, processing the stool sample, and sequencing the microbiome of the individual are performed at least twice ¨
once before the individual acquires a health condition (known as a baseline) and at least once alter the individual acquired the health condition. This is necessary so that the baseline microbiome can be compared to the microbiome when the individual is suffering from a health condition.
[00183] Optionally, the steps of acquiring a stool sample, processing the stool sample, and sequencing the microbiome of the individual are performed for a third time, after the individual has overcome the health condition, to confirm that the individual is healthy again.

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [001841 When the assay shown above was tested on multiple individuals, the following organisms were detected as part of the assay: Bacteroides Eragilis, Clostridioides Escherichia coli. The most abundant organism was Bacteroides fragilis (8.10%).
and the mean abundance of the detected organisms was 2.87%. The total number of reads in the sample was 26,012,172 1001851 Based upon phylum, the most abundant organisms were.
Bacteroidetes at 80.90%, Firmicutes at 16.72%, Proteobacteria at 1.95%, Actinbacteria at 0.43%, Verrucomicrobia at 0.00%, Ascomycota at 0.00%, Candidatus Saccharibacteria at 0.00 %, Fusobacteria at 0.00%, and Basidiomycota at 0.00%.
100186] Based upon class, the most abundant organisms were:
Bacteroidia at 80.90%, Clostridia at 15.49%, Betaproteobacteria at 0.99%, Deltaproteobacteria at 0.60%, Etysipelotrichia at 0 47%, Negativicutes at 0.41%, Oammaprotwbacteria at 0.36%.
Coriobacteria at 0.29%. Actinobacteria at 0.15%, and other at 0 35%.
1001871 Based upon family, the most abundant organisms were:
Bacteriodaceae at 74,50%, Ruminococcaceae at 4.09%, Tarmerellaceae at 3.32%, Rikenellaceae at 2 80%, Clostridiaceae at 2 12%, Lachnospiraceae at 1.99%. Eubacteriaceae at 1.83%, Sutterellaceae at 0.91%, Peptostreptococcaceae at 0.63% and other at 7.80%
1001881 Based upon species, the most abundant organisms were.
Bacteriodes uniformis at 56,89%, Bactertodes fragilis at 8.10%, Bacteriodes stercot is at 5.35%, riacteroides stercoris CAO:120 at 4%. Clostridiales bacterium at between 4% arid 3.3%, Parabacteriodes merdea at 3.32%, Faecalibacterium prausnitzil at 2.58%, Altstipes ptitredinis at 1.32%, lEubacteriumj hail at 1.08%. and other at 13.78%
[001891 The present invention also comprises a screening kit or assay that screens for the above listed 48 organisms PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [001901 By screening for the above listed organisms, different diseases and conditions can be determined, such as: Autism, Crohn's disease, Chronic Urinary Tract Infections, Clostrldoides dIffictle infection, Obesity, Alzheimer's disease, Psoriasis, Dietary Impact, Mylagic Encephalomyelitis/Chronic Fatigue Syndrome, the effect of diet, and COVID-19.
See Appendix' B-M for the protocols related to these diseases/issues.
1001911 By applying the above procedures and screening for the 48 organisms listed above, it was determined that.
100192] It is essential to compare the microbiomes of mother to child, sibling to sibling, and/or disease within disease;
1001931 Although everyone is an individual, each individual has a different microbiome;
1001941 A biological child an mother is initially born with the same trucrobiome of the mother;
1001951 Within families of individuals, there is a similarity in the microbiome's between those familial individuals, however, people that are not related are not completely different;
1001961 Within diseases, there is a similarity in the microbiome of individuals that suffer from the same disease;
1001971 'There is a loss of diversity of the microbiome in individuals with Crohn's disease and autism;
[001981 It is helpful to compare within the family or within the individual (baseline vs.
disease, or disease vs. cured);
1001991 Toxoplasma gondii is a commonality found within patients with Crohn's Disease;
100200] Loss of diversity was noted in children as compared to mothers;

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [00201] In order for an individual to avoid getting Clostridium difficile. the individual needs multiple families of clostridiums within their gut. For an individual to avoid having the plague, the individual needs multiple families of Yersinia within their gut;
100202] Clostridium difficile is present in everyone and Clostridium difficile generic testing is better than what is currently being utilized to test for Clostridium difficile;
100203] Not all Crohn's Diseases are the same There are different organisms that are involved that cause different versions of Crohn's Disease;
1002041 Obtaining a baseline from patients when they healthy and comparing that baseline to when they start developing a disease is important;
1002051 Sequencing the microbiome of a biological mother and a biological child, analyzing the differences between the two of them, and then comparing the differences between mother and child to other patients with the same disease showed that there was a difference in the organisms between the mother and child, and the microbiome varies from individual to individual. The child was then evaluated to determine what organisms the child was missing and the mother was then evaluated to determine what organisms the mother was missing, and the missing organisms from the mother and the child were then compared. It was noted that within families there is the same pattern of microbes (missing versus present);
and High clostridiums bacteroides and staphylococcus are a marker of Celiac sprue.

[002071 Emunpieli Crohn's Disease 100208] Crohn's Disease (Cl)), a serious, potentially life-threatening, and debilitating condition which usually affects children, teenagers. and young adults, is an inflammatory bowel disease with a typical age of onset between 15 and 25 years of age.
Symptoms can include pain, diarrhea, and other intestinal problems. CD appears to show some familial predisposition, as approximately 20-30% of people with CD have a direct blood relative with PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attoiney Docket No. 2042553 some form of IBD. Men and women are equally affected. The objective of this example is to determine the dysbiosis conditions under which Crohn's disease develops.
[002091 The following procedure was completed on 19 patients suffering from Crohn's disease. Shotgun Sequencing was performed. Shotgun sequencing is a laboratory technique for determining the DNA sequence of an organism's genome. The method involves breaking the genome into a collection of small DNA fragments that are sequenced individually. A
computer program looks for overlaps in the DNA sequences and uses them to place the individual fragments in their correct order to reconstitute the genome.
[002101 More specifically, patient stool samples were collected utilizing collection vials.
Following fecal collection, individual patient DNA was extracted purified with a DNA
extraction kit. The isolated DNA was then quantitated utilizing a fluorometer.
1002111 After DNA quantification, the DNA was normalized and the library was prepared. This process utilized the shotgun workflow wherein the samples underwent tagmentation, purification, amplification and indexing, followed by a final purification step.
[00212j Samples libraries were then normalized and combined to create a library pool which was quantified and appropriately diluted to the final loading concentration to be sequenced on the appropriate DNA sequencing system/machine.
1002131 Once the DNA sequencing was complete, the raw.bc1 data was converted to FASTQ files. The FASTQfiles were then pushed through the bioinformatics metagenomics pipeline with patient specific endpoint readouts profiling each individual's unique microbiome.
1002141 More specifically, the bioinformatics pipeline utilized a computational tool that profiled the microbial communities from metagenomic sequencing data with species level resolution. Patient microbiome profiles were then analyzed to ascertain not only the profile of PCT/u520/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 microbes in the patient samples but also to identify specific strains, and provide accurate estimation of organisml abundance relative to the overall diversity.
[00215] Additionally, patient specific microbiome profiles were aligned and compared to their medical records and other patient provided information for further analysis and interpretation.
1002161 The patient sample was stored for future use in a 20 C freezer.
1002171 Table 4 documents organisms that were discovered in each of the 19 patient samples. The first row of Table 4 contains the Patient ID numbers. which are represented throughout the Figures and 'fables.
[Clostridium] L 1 1 I I I 1 1 1 1 1 1 bollette ___________________ JClostridiuM) 1 1 u 1 1 1 1 0 1 1 1 seindens [Clostridium] 1 1 1 1111 t 1 saccharolvt icun [Clostridium] 1 1 1 I 1 1 1 0 1 1 1 .s2henoides [Clostridium] 1 1 1 1 1 1 0 1 1 I 1 Clostridiales i 0 1 I 1 1101 baclenum Closiridiales 1 0 1 I I I I 0 I 1 1 bacterium Clostridium 1 1 1 1 1 1 1 0 1 1 1 sporogenes Clostridium 1 1 0 1 1 1 1 0 1 1 1 sp. SY8519 Clostridium 0 1 1 1 0 1 1 1 1 1 1 botulinum 202F
Clostridium 1 1 1 1 1 1 1 0 1 1 1 0 1 botulinum 13 Clostridium 0 U 0 0 0 0 1 () 1 1 1 1 1 botulinum BK11)15925 -elostridi tun 0 0 1 1 1 0 0 () 1 0 1 0 1 0 1 1 ¨ 0 0 bottilinum A2_ _ Clostridium 7-7¨i11114)111 1 1 1 1 1 1 1 ¨1 1-$7.
_Witt limn Al 'Clostridium Tit io I ______________ 0 bolulintint B1 ;-____________________________________________________________________________ =
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION Attorney Docket No. 20-42553 Org. Name cont. A B C I) I - 1 2 3 4 5 6 7 )4 9 10 11 12 13 14 T
Clostridium TTTTT I 0 1 1 1 1 I-1 1 I 1 0 0 161 bolulinum F
Clostridium 1 1 I 1 1 1 0 1 1 1 1 botulinum Clostridium 0 1 1 1 I 0 0 0 1 I 0 botulinum , CDC 1436 Clostridium -70 1 0 0 0 0 0 0 1 0 1 0 0 0 1 I 0 0 5 botillinum E3 Clostridium 0 0 0 0 0 0 0 0 1 1 1 I I 0 0 1 1 I 0 8 bohtlinuin PteV0( 394 Clostridium I 1 1 1 I 1 1 1 1 0 1 1 I I I 1 I 1 18-i perfringens Clostridium 0 1 1 1 1 1 0 0 1 1 1 1 1 1 0 1 1 0 1 14 Perfringens F262 Clostridium 0 1 1 1 1 I 0 0 I 1 1 porfringens ATCC
Clostridium 0 0 0 0 0 1 1 0 1 1 - 1 I
1 1 1 1 1 0 o 11 perfringens str. 13 Clostridium 0 1 1 0 1 1 1 1 1 1 1 3_ Clostridium I 0 -7 1 1 1 1 0 1 1 1 beijerinekii NCNB
Clostridium = 1 0 0 1 7¨ 1 I 0 1 1 1 I
F 1 1 15-1.' 1 1 1 tic ijerinokii NRRL
Clostridium 1 7 1 1 1 1 1 1 1 ¨I
1 1 1 ITi 19-1 butyricum Clostridium 0 I I 1 1 1 1 __ 1 1 1 baratii Clostridi tun 1 1 1 1 1 1 1 0 1 I 1 sp. CT4 Clostridium 1 1 1 1 I I 1 0 I 1 I

pastourianum BC I
Clostridium 1 0 0 1 0 0 1 0 1 1 1 burmii sir.
Clostridium 1 1 1 1 I 1 1 0 1 1 1 isatidis Clostridium 1 0 1 0 1 1 1 0 1 171 I

Jicelotnuxycut9 _ _ e53.;Fri-diuni '5-7 7- 1 I (-7 -6 0 -5 ¨a I 0 0 F.;
kluyveri 1)SM 355 Clostridium 1 1 0 1 1 1 0 1 I I 1 1 1 1 1 1 I7 1 17 sp. DL-Vill Clostridium 1 I 1 1 i 1 1 1 I 1 1 0 acetiCUM
Clostridium 1 1 1 1 I 1 1 1 1 1 1 1 septictun Clostriditun 1 0 0 1 0 1 1 0 1 I 1 1 itoµyi NT

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION Attorney Docket No. 20-42553 Org. Name Cunt, A B C D E 1 2 3 4 5 6 7 N 9 10 ¨11 12 13 14 T 1 Clostridiuni- ¨ -1 --i¨ 1 0 i 0 0 0 0 1 1 1 1 1 1 1 1-- 1- ' 7 ¨14 cellulovorans 74313 , - i - _ Clostridium 1 1 1 1 1 0 1 1 1 I 1 1 1 I
argentinense . .
C lostri di um 1 1 1 I 1 I 0 0 1 1 1 1 bomimense Clostridium 1 I 1 1 1 1 1 1 1 I 1 1 I

sp. BNL1100 Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1 cochlearium Clostridiwn 1 1 1 1 1 1 1 0 1 1 1 1 1 sp. JN50090 I _ , _ . . , , Clostridium 1 1 1 1 1 1 1 0 I 1 1 I 1 sp. IN-9 Clostridium 1 I 0 I 1 1 0 (I I 1 I 0 1 sp. JN-I . . _ Clostriditun 1 I 1 1 1 1 0 1 0 1 1 1 1 Saccharo=
. butylicum 1 , , , , , , , Clostridium 1 1 1 1 1 1 1 0 1 1 1 I 1 , tyrobutyricum _ _ .
Clostridium 1 1 1 1 1 1 1 1 1 1 1 1 1 1 estertheticum ...... , Clostridium 1 1 1 1 1 ' 1 I -0 1 ' 1 1 0 1 1 ' 1 - 1 1 I 1 17 Carboxidivorans Clostriditun 1 1) 1 1 1 1 1 1 I I 1 1 1 l 1 1 1 1 1 18 jomucaceticurn Clostri di in 1 I 1 (1 1 1 1 1 1 1 1 1 chauwei Clostridium I I) 0 1 I 1 1 1 1 1 0 1 1 Clostridium 1 1 1.1 I 1 1 1 1 I 1 1 I 1 tetani Clostridiun) 1 I 1 1 1 1 1 0 1 1 1 1 1 !Wain Clostridium 1 1 1 1 1 1 1 0 1 I 1 1 1 tenni E88 Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1 scatologenes , Clostridium 1 0 1 1 1 1 0 0 1 1 1 1 1 tueniosporum . . ¨ . .
Clostridium 1 I I 1 1 1 1 1 1 1 1 1 1 drake] I .
Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1 autoethanogenum Clostridium 1 0 0 0 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 14 sp. MF28 .
Clostridium 1 1 1 1 1 1 1 0 1 1 1 1 1 ljungdallIti DSM
Clostridiaceae 1 I 1 1 1 1 1 0 0 1 1 bacterium Clostridioides 1 1 1 1 1 1 1 1 I 1 1 dilEcile . _ _ PC17us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION Attorney Docket No. 20-42553 Org. Name Cont. A B C 1) E 1 2 3 4 ¨5 ¨6 7 8 9 10 11 12 13 Claitridioides 1 1 1-1 1 1 1 0 1 1 1 1 difficile ATCC
C lost ridioldes 0 0 0 0 0 I 1 0 1 1 1 ditilcite Q.CD.63q42 Clastridioicics 1 1 U 1 1 1 0 0 1 1U 1 0 1 0 0 1 1 0 11 difficile M120 Clostridioides 0 0 0 0 0 1 7-- 1¨
_______________________ 1 0 3 o 1 1 0 1 9 OCI)-37x79 Clostridloides 0 1 1 0 1 0 0 0 0 1 1 1 0 I 0 0 0 0 0 7 difficilo M68 Clostridioides 0 0 1 1 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 5 difficile 630 Clostridioidos I I 0 0 I 0 0 0 1 1 0 I 0 0 0 0 I 0 0 7 difficilc OP
Clostridioldos 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 0 0 0 3 difficila CD196 Clostridloides 0 0 0 (1 0 0 0 0 0 1 0 0 0 0 0 1 1 0 0 :T.¨I
diflicile QCD-76w55 Clostridioides 0 0 0 0 0 0 0 0 0 0 0 0 1 Cl (1 0 l 0 1 , 3 difficile OCD-66c26 , ______________________________________________________________________________ (Clostridium] 1 0 1 0 1 I 1 0 1 1 1 innocuum 'Clostridium' 1 0 1 0 1 1 1 0 0 1 1 ultuncuse Clostridium 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 formicncoticum _ Clostridium 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 sp. AWRP
Clostridiales 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 3 bacterium Closuidialos 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 3 bacterium [Clostridium' 0 1 0 1 0 1 0 0 1 0 0 0 0 0 0 (1 0 1 1 6 ultunense Lisp Clostridium 0 0 1 1 0 0 1 1 1 1 0 I

kluyvori Clostridium 0 0 0 1 ¨0 1 1 1 1 0 0 0 1 0 0 0 1 0 7 collulovorans Clostridium Saccharoper-bulylacetonicum Total 59 59 57 60 64 65 56 26 70 75 69 70 70 1 70 59 70 76 66 Clostridia Tecies Table 4 =
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 1002181 Table 5, shown below, documents the total numbers of the different species of bacteria/organisms present in all 19 patient samples combined. The data documented in Table is shown in Figure 4.
beies Number PiTsertt_ Clostridioides difficile 19 Clostridioides difficile ATCC 18 Clostridioides difficile QCD-_61.9.1_4.2 .....
Clostridioides difflcile M120 11 Species Coot. Number Present Clostridioides difficile QCD-37x79 9 Clostridioides difficile M68 7 Clostridioides difficile 630 5 Clostridioides difficile CIP 7 Clostridioides difficile CD196 3 Clostridioides difflcile QCD=
76\05 3 Clostridioides difficile OCD-66c26 3 Table 5 [00219} Table 6 documents the mycobacterium found in the samples.
Organism 1 6 9 11 2 3 5 10 12 13 14 Total Organism name Name Myco 1 I 1 1 I 0 1 1 1 0 I 5 Salmonella enterica bacterium Myco 1 I 1 1 0 0 1 0 1 0 I 3 Salmonella enterica bacterium subsp.
enterieu clumaera serovur Brancaster Mycobacterium 1 1 1 1 0 0 1 1 1 0 1 4 Salmonella enterica intracellulare nibs?.
cntcrira subsp. I SCTOVII r Chester Yonzonense Myco 1 0 1 1 1 1 1 1 1 1 1 7 bacterium mum Mycobacterium 1 0 1 1 0 1 1 0 0 0 0 2 Salmonella enterica avitun subsp. subsp.
enterica Parutubentdosis serovar Minnesota__ Mycobactetium 0 1 I 1 1 0 1 0 0 0 1 3 subsp.
hominissuis Mycobiteteriunt 0 1 0 1 0 1 0 0 0 0 1 avium 104 , . . .
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 Org, Name 1 6 9 11 2 3 5 [10 12 13 14 Total Organism name Cont. -I¨ ¨
Mycobacterium I 1 1 1 I 0 1 1 1 1 0 1

5 Salmonella erueric¨a ¨
marscillensc subsp.
enterica scrovar Macclesfield ¨
Mycobacterium I 1 I 1 0 0 1 0 1 0 1 3 Salmonella enterica lepmernuriuni subsp.
enterica --i--- 4 serovar Tennessee =-1 Mycobacterium 1 I 1 I 0 0 I 0 0 0 0 1 Salmonella enterica paraintracellulare subsp.
enterica serovar Rubislaw ,.
Mycobacterium 1 1 I I 1 0 1 0 I I 1 5 Salmonella entcrica sp. EPa45 subsp.
enterica wow Typhitnurium , Mycobacteritun 1 1 1 1 1 0 1 1 1 1 1 1

6 Salmonella enterica sp. YC-RIA subsp.
enterica serovar Senflenbele,.
¨
Mycobactenum I 1 1 I 1 0 1 1 1 1 1 6 Salmonella enterica sp. MS1601 I subsp.
entcrica serovur Waycross ...
Mycobacterium 1 1 I I 1 0 1 10 1 1 1 5 Salmonella entcrica dioxanotrophicus subsp.
enterica serovar Weltevreden Mycobacteritun 1 1 1 1 0 0 1 0 1 0 1 3 Salmonella cnterica sp. VKM Ac- subsp.
enterica 18171) serovar Choleracsuis Mycobacterium 1 1 1 1 1 ' 0 1 1 1 0 1 5 Salmonella cnIerica kansasit subsp enterica serovar Samtpaul , Mycobacterium I I 1 1 1 0 1 0 1 I 1 5 Salmonella enterica sp. 0-10 subsp.
enterica SCTOVilt Stanley Mycobacterium 1 I I 1 1 0 1 0 1 0 1 4 Salmonella enterica sp. JS623 subsp.
cntcrica SefOVIIr Apapa Mycobacterium 1 I 1 1 1 0 I I 1 0 1 5 Salmonella enteric(' lepme subsp.
enteric(' ___________________________________________________________________________ setavar Djakarta __...
- _______________ Mycobacteritun 1 I 0 1 1 1 1 0¨ 1 0 0 4 Salmonella enteric(' shigtiense subsp.
enterica serovar Albany Mycobacteritint 1 1 1 I 1 1 1 I 1 1 1

7 Salmonella enterica sp. DL90 subsp.
entcrica serovar Milwaukee Nlycobacterittni 1 1 1 1 U 0 1 0 0 0 0 I
Salmonella enterica canettii CIPT subsp.
enterica 1.10070011) =rover Thompson _.
Mycobacterium 1 I 1 1 0 0 1 0 1 0 0 2 Salmonella anerica caneilii CIPT subsp.
entcrica 140070017 ....1 _________________________________ serovar Stanislyi I lc _ Mycobacteri am 0 0 0 0 0 0 1 0 1 0 0 2 =wail CIPT
140070008 , Mycobacterium 0 1 0 0 0 0 0 0 0 0 0 0 cant i OPT

I

PCT/us20/44605 26 May 2023. (26.05.2021) PCT APPLICATION
Attorney Docket No, 20-42533 Org. Name 1 6 9 11 2 3 5 10 12 13 14 Total Organism name Cont.
Mycobacterium 1 1 1 1 1 0 1 0 1 1 1 5 Salmonella enterica tuberculosis subsp. salamac scrovar 55:k:z39 Mycobacterium 1 1 1 1 1 0 1 0 1 0 1 4 hacmophilum Mycobacterium 1 1 1 1 1 0 1 0 1 0 1 4 haemophilum Mycobacterium 1 1 1 1 1 0 1 1 1 0 1 sp WY10 MycobaTiirtrin- T I ¨1 1 ¨0 1 1 0 1 1 1 5 paragordonae Mycobacterium 1 1 I 1 1 0 1 0 1 1 1 3 marinum Mycobacterium 1 1 1 1 1 0 1 .11 1 1 0 5 sp. JLS
Mycobacterituti 1 1 1 1 1 0 1 0 0 0 0 2 ¨
sp. TTR15 Mycobacterium I I 1 0 0 0 1 0 1 0 0 2 ulcerous subsp, shinshitense Myeobactcriten 1 O. 1 1 1 0 0 1 1 0 0 3 liflandii 128FXT
Mycobacterium 1 1 1 1 0 0 0 0 1 0 1 2 sp. Q1A-37 [Mycobacterium] 1 1 1 1 0 0 0 0 0 0 0 0 stephanolepidis I.MYeobacteriu to] 1111 0 0 0 0 0 0 0 cbelonae subsp (iwaiuikae MyCObtleteriUITI 0 0 0 0 U 0 1 I 0 0 0 0 1 sp. MOTT36Y
Mycobacterium 0 0 0 0 0 0 0 0 0 0 1 1 pseudoshottsli 34 14 34 35 22 5 34 12 29 11 25 19.7 Table 6 1002201 .. Figure 5 is a graphical representation of the biodiversity of mycobacterium in healthy patients versus patients Nt.ltil Cran's Disease. Crohn's patients are shown using the solid black bars and healthy patients are shown using the series of smaller black bars 1002211 Figure 6 is a graphical representation of the mycobacterium of patient 12 compared to patient 12's biological mother (patient I 1).
(002221 Figure 7 is a graphical representation of mycobacterium of patient 2 compared to patient 2's biological mother (patient 1).

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION Attorney Docket No. 20-1002231 Figure 8 is a graphical representation cube mycobacterium of patient 10 versus patient 10's biological mother (patient 9).
100224! Table 7, shown below, documents the possible causes of Crohn's disease.
Osganisin Name 1 6 9 11 Total Toxoplastna gondii ME49 1 1 1 I 4 Bacteroides fragilis 1 1 1 1 4 Bacteroides fragilis 638R 1 1 1 , 1 4 Organism Nitnte Coot, 1 6 9 11 Total Bacteroides frailis YCH46 1 1 1 1 4 Bacteroides fragilis NCTC 9343 1 1 1 1 4 Helicobacter hepaticus 1 1 1 1 4 Helicobacter hopaticus ATCC

Table 7 (00225) Table

8, shown below, documents the possible causes of Crohn's disease.
Organism Name 2 3 5 10 12 13 14 Total Toxoplasma gondii ME49 1 1 1 1 1 1 I

Bacteroides I 7 Bacteroides fss 638R ____________________ 1 0 1 1 1 1 1 Bacteroides fragilis YCH46 1 0 1 1 1 I 1 Bacteroides fragilis NCTC 9343 1 0 1 1 1 1 1 6 Helicobacter hepaticus 1 0 1 0 1 1 1 Helicobacter he.pliticus ATCC 51449 I0 1 _!.L I 5 Table 8 [00226) Table

9, shown below, documents the possible causes of Crohn's disease.
. Organism Name 1 6 9 11 Total Yersinia enterocolitica 1 111 1 4 Yersinia similis 1 I 1 1 4 Yersinia pscudotuberculosis 1 I 1 1 4 Yersinia_pestis 1 1 , 1 1 4 ...Yeis.iniaps.tis 1.1 0._. 0 Yersinia pestis Angola 0 0 1 0 0 0 Yersinia pestis str, Pestoides B 0 1 0 0 I
Yersinia pest is 3770 0 0 , 0 0 , 0 , . . .
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION Attorney Docket No. 20-Organism Name Cont. 1 ..LL 11 Total Yersinia pestis 2944 0 0 0 0 0 Yersinia pestis 790 0 0 I 0 0 0 Yersinia pestis 1045 0 0 0 0 0 YersiniaTestis D182038 0 0 0 0 0 Yersinia entomo_phap 1 1 1 1 4 _ Yersinia ruckeri I 0 I 1 3 Yersinia frecleriksenli 1 1 1 1 4 Yersinia rohdei 1 1 1 1 4 Yersinia aldovae 670-83 1 1 1 1 4 Yersinia aleksiciae 1 1 1 1 1 4 I
Yersinia sp. CFS1934 I 1 1 1 1 4 Yersinia massillensis 0 I 0 I -, Yersinia kristensenii 0 0 0 1 1 --Yersinia intermedia C1 I 0 0 1 12 14 11 13 12.5 Table 9 1902271 Table

10, shown below, documents the possible causes of Crohn's disease.
Organism Name 2 3 5 10 12 . 13 14 Total Yersinia enterocolitica 1 1 1 1 1 1 1 7 .
Yersinia similis 1 1 I 1 1 1 I __ 7 _ Yersinia pseudotuberculosis __________ _L 1 1 1 1 7 ¨
Yersinia pestis 1 1 1 0 1 1 , 1 Yersinia pestis Antigua () 0 I 0 , 0 0 0 Yersinia pestis Angola 1 0 1 0 0 0 0 /
_ Yersinia,pestis str. Pestoides B 1 0 0 0 0 0 0 1 Yersinia pestis 3770 0 1 0 0 0 1 0 , Yersinia pestis 2944 0 1 1 0 0 0 0 .-) Yersinia pestis 790 0 0 I 0 0 0 0 1 Yersinia pestis 1045 0 0 0 0 0 _ 1 0 1 .
Yersinia pestis D182038 0 0 0 0 1 0 0 4_ 1 _ . Yersinia entomoplAga 1 1 1 1 1 1 1 1 Yersinia ruckeri 1 1 1 1 0 1 1 6 Yersinia frederiksenii 1 1 I 1 1 1 1 7 _ Yersinia rohdei 0 I I 0 1 I 1 5 Yersinia aldovae 670-83 1 1 1 1 1 1 1 Yersinia aleksiciae 0 0 1 0 1 1 1 4 Yersinia sp. CFS1934 0 1 1 1 1 1 0 5 Yersinia massiliensis 1 0 1 0 1 0 0 3 .
Yersinia kristensenii 0 1 1 __ 0 0 0 0 2 - . _ Yersinia intermedia 0 0 1 0 1 0 0 2 = =
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553

11 13 I 18 L 8 L 13 [ 13 I 10 12.2857143j Table 10 [002281 Figure 9 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother (patient 11).
[00229] Figure 10 shows a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother (patient 11).
[00230] Figure II shows a graphical representation of a comparison of the microbiome between patient 2 and patient 2's biological mother (patient 1).
[00231] Figure 12 shows a graphical representation of a comparison of the microbiome between patient 2 and patient 2's biological mother (patient 1).
[00232] Figure 13 shows a graphical representation of a comparison of the microbiome between patient 14 and patient I4's biological brother (patient 6).
[00233] Figure 14 shows a graphical representation of a comparison or the microbiome between patient 10 and patient 10's biological mother (patient 9).
[00234] Table 11. shown below, documents common organisms found in patients with Crohn's disease.
FAMILIES
FT-0001-500ng s Tox2p1asma end ii 59 FT-000I
FT-0002 s To xo.plasma. gpndii 36,278 FT-0002 FT-0006 s Toxoplasma gondii 68 FT-0006 Fr-ooi 4 s l'oxoplasma gondii 14,312 FT-0014 FT-0009 s Toxoplasma gondii , 32 FT-0009 PT-0010 s Toxoplasma gondii 31,855 FT-0010 FT-0011 s Toxoplasma gondii 52 FT-0011 FT-0012 s Toxo_plasma gondii 1,425 FT-0012 Table 11 100235] Table 12, shown below, documents common organisms found in patients with Crohn's disease.

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Attorney Docket No. 20-42553 CHRON___ F1'-0002 s ToxoplasmEt gondii 36,278 F1-0003 s Toxoplasma gondii 19,625 FT-0005 s Toxoplasma gondii 206 FT-0010 _ s To:.(2plasinIzondii 31,855 FT- oo s Toxoplasmajlondii 1,425 FT-0013 sToxoplasma gondii 22,864 FT-0014 s_Toxoplasma gondii 14,312 Table 12 [002361 Figures 15, 16 and 17 are graphical representations of common microbes round in patients with Crohn's disease. tyle_mmeificlitY.....F.1111,a...15....09.ws th1.11/TaUnt Qf bacteroide4.
frggilis foadjazdentsikthadja:s disease as ssanpajesi to healthy Camity members. and Eigurgalfand..1111-KAutig.,:ding.v.nlisfigaituraa..gostiiloartip patientuyith_Culin's 1115.e.1%.
1002371 Table 13, shown below, documents common organisms found in patients with Crohn's disease.
FAMILIES
FT-QUO 1-500ne s Escherichia coli 603 FT-0001 FT-0002 s Escherichia coli 239.346 FT-0002 FT-0006 s Escherichia coli 121,584 FT-0006 FT-0014 s Escherichia coli 6,501 Fr-0014 FT-0009 s Escherichia cob 486 FT-0009 FT-0010 s Escherichia coli 174,401 FT-0010 FT-0011 s Escherichia coli 405 FT-0011 FT-0012 s Escherichia coli 1,589 FT-0012 Table 13 [002381 Table 14, shown below, documents common organisms found in patients with CrOhnsS disease.
CEIRON
FT-0002 s Escherichia coil 239 346 FT-0003 s_Escherichia coli 31,16,1 FT-0005 s _Escherichia coli 330,582 FT-0010 s Escherichia coli 174,401 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No, 20-42553 CH1R0p1 Copt.
FT-0012 s Escherichia coil 1,589 FT-0013 s Escherichia coil 91,329 , FT40014 s Escherichia coil 6,501 Table 14 [00239] Figure 18 is a graphical representation showing the amount of Eschorichuz coil eatiinien-efttal4944-15 found in patients with Crohn's disease.
1002401 Figure 19 is a graphical representation showing the amount of Escherichicz colt common orgnisni found in patients with Crohn's disease (shown with the solid black bars) and healthy family members of those patients (shown with the series of solid black bars).

Table 15, shown below, documents common organisms found in patients with Crohn's disease.
FAMILIES
FT4X)01-500ng s_Bacteroides fragilis 86,801 FT-0001 FT-0002 s_Bacteroides fragilis 6,461 FT-0002 ST-0006 s_13Qcteroides fragilis 56.124 FT-0006 FT-0014 s Bacteroides fragilis 33,504 FT-0014 FT-0009 s Bacteroides fragilis 63,219 ; FT-0009 FT-0010 s Bacteroides fragilis 4,636 FT-0010 1T-0011_ Bacts.roides fiagjlis 75 FT-0012 s Bacteroides fragilis 1,382,505 FT-0012 Table 15 Table 16, shown below, documents common organisms found in patients with Crohn's disease.
Cl-IRON
..... ¨
FT-0002 s Bacteroides fragilis 6.461_ FT-0003-500ng s Bacteroides fragilis 4 FT-0005 s_Bacteroides fragilis 54,107 FT-0010 s_Bacteroides fragilis 4,636 FT-0012 s Bacteroides fragilis 1,382 c05 FT-0013 s___Bacteroides fragilis 31,886_, FT-0014 s _Bacteroides fragilis 33,504 Table 16 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [00243] Figure 20 is a graphical representation showing the amount of bacteroides fmilts einrneegisms iblUld in patients with Cretin's disease (shown in solid back bars) as oromrslia_lanlbkually..inuotga ______ (sho.vii viallffiOS.Ligag.U2411).
[00244] Table 17, shown below, documents common organisms found in patients with Crohn's disease.
FAMILIES
FT-0001-500ng s Mycobacterium avium 40 FT-0002 s Mycobacterium Mit=
FT-0006 s My9.9bacterium 35 FT-00I4 s Mxcobacteriutn avium 6 FT-0009 s Mycobacterium avium 28 FT-0010 s Mycobacterium avium FT-001 I s Mycobacterium avium 56 FT-0012 s Mycobacterium awl= 3 Table 17 [00245] Table 18, shown below, documents common organisms found in patients with Crohn's disease CHRON
FT-0002 s Mycobacterium avium 1 FT-0002 .FT:0003-500ra___, s MIcobacterium.ayium 2 FT-0003_ FT-0005 s Mycobacterium avium 54 FT-0005 FT-0010 s Mycobacterium avium 2 FT-0010 FT-0012 s Mycobacterium avium 3 FT-0012 FT-0013 ........................ s Mycobacterium avium 4 FT-0013 FT-0014 s Mycobacterium avium 6 FT-0014 Table 18 [00246) Figure 21 is a graphical representation showing common organisms found in patients with Crohn's disease. More_socifically. Figureal_ shows Crobrts Patients with AziYaglg.cLeti.wn iANIiin..:subWecieLZM4111.12grekIlosis [00247] 'fable 19. shown below, documents common organisms found in patients with Crohn's disease.

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Attorney Docket No. 20-42553 FAMILIES
FT-0001-500ng s Helicobacter hepaticus 36 FT-0002 s Helicobacter hepaticus 1 FT-0006 s Helicobacter hepaticus 18 FAMILIES Cont.
FT-00I4 Helicobacter heaticus _ FT-0009 s Helieobacter hepaticus 69 FT-0010 s_Helicobacter hepaticus 0_ FT-0011 s.,,..Helicobacter hepaticus 2 yr-ool2 s Helicobacter hspaticus 5 Table 19 j002481 Table 20, shown below, documents common organisms found in patients with Crohn's disease.
CHRON
FT-0002 s Helicobacter hepaticus 1 FT-0003-500ng s Helicobacter hepaticus 0 FT-0005 s Frei icobacler hepaticus FT-0010 s__Helicobacter hgaticus 0 FT-0012 s Helicobacter hepaticus 5 FT-0013 s Helicobacter hepaticus 9 FT-0014 s Helicobacter hepaticus 3 Table 20 100249]
Table 21, shown below, documents common organisms found in patients with Crohn's disease.
FAMILIES
FT-0001-50ons s Enterococcus faecalis 36 FT-0002 s Enterococcus faecalis 445 FT-0006 s Enterococcus faecalis 150 FT-0014 s _Enterococcus faecalis 31 FT-0009 s5 Enterococcus faecalis 150 FT-0010 s Enterococcus faecalis 20 FT-0011 s Enterococcus faecalis 247 FT-0012 s Enterococcus faecalis 193 Table 21 . , .
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 (002501 Table 22, shown below, documents common organisms found in patients with Crohn's disease.
CHRON
FT-0002 $ Enterococcus faecalis 36 FT-0003-500ng s Enterococcus faecalis 0 _ FT-0005 s Emerococcus faecalis 501 FT-0010 .._ s Enterococcus faecalis _ _ _ 20 FT-0012 s Enterococcus faecalis 193 FT-0013 s_Enterococcus faecalis 58 FT-00I4 s____Enterococcus faecalis 31 Table 22 (00251.) Table 23, shown below, documents common organisms found in patients with Crohn's disease - _______________________________________________________________________ ,Patient ID 1 2 11 12 14 _ 6 Enterococcus faecalis V583 1 1 1 1 , 1 1 Enterococcus faecalis D32 0 0 1 L 1 1 Enterococcus faecalis AROliDG 1 0 1 0 0 1 Enterococcus faecalis DENG1 0 0 1 1 0 0 Enterococcus faocalis A'TCC 1 0 1 0 1 1 _ Patient ID Cont. 1 2 11 12 14 6 . _ Enterococcus faecalis str Symbiaflor 1 1 0 1 1 0 0 Mycobacterium minim subsp.
paratuberculosis I 0 1 I 0 0 Malassezia furfur 0 () 0 0 0 0 Table 23 100252) Figure 22 is a graphical representation of a comparison of the microbiome between patient I and patient I 's biological mother (patient 2).
[002531 Figure 23 is a graphical representation of a comparison of the inicrobiome between patient 12 and patient 12's biological mother (patient 11) 1002541 Figure 24 is a graphical representation of a comparison of the inicrobiome between patient 2 and patient 2's biological mother tpatient I ).

PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [00255] Figure 25 is a graphical representation of a comparison of the microbiome between patient 14 and patient 14's biological brother (patient 6).
[00256] Figure 26 is a graphical representation of a comparison of the microbiome between patient 12 and patient 12's biological mother jpatient 11).
[00257] Figure 27-29 are graphical representations showing common organisms found in patients with Crohn's disease. Meremeciti.e 1 FaiL_IgurellshoEuLoomparison of the amount of enterococcus faecalis found in patients with Crohn's disease (shown via the darker bars) and healOw family members (shown via the lighter bars), [00258] Figure 28 shows a comparison of the amount of helicobacter heotaticus found in patients with Crohn's disease (shown via the lighter brut and healthy family members (shown via the darker bars).
[00259] figure 29 shows a comparison of the amount of texonlasma Rondii found in patients witb Crohn's disease (shown via the lighter bars) and healthy family members (shown via the darker bars. but the darker bars are near zero and are difficult to view) [00260] F,xample Chronic Urinary Tract Infection [00261] Chronic urinary tract infections (UTIs) are painful and frustrating for patients The symptoms of a lower urinary tract include frequent and/or urgent need to urinate, dysuria, soreness in the lower abdomen, back, or sides, pain on urination, need to urinate at night, and urine that is discolored potentially with a foul odor. If the infection is in the kidneys it can be life threatening. There are many proposed causes of chronic ur Is, however some studies have indicated that dysbiosis of the gut microbiome may play a role. The objective of this example is to analyze the micro biome of patients with chronic UTIs to look for similarities in relative abundance of microbes and groups of microbes [00262] The same procedure noted above for Example 1 was performed on 30 individuals suffering from chronic urinary tract infection.

=
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 1002631 Emop10. Clostridoides difficile Infection 1002641 Clostridoides difficile is a gram-positive spore-forming rod-shaped bacterium which can cause severe illness. Infection with C. difficile frequently occurs following antibiotic use, suggesting that dysbiosis, or an imbalance of the microbiome of the gut, could play a major role in the development of infection. The obectvie of this example is to correlate conditions in the microbiome which could contribute to, or be the result of, infection with C.
[002651 The same procedure noted above for Example 1 was performed on 30 individuals suffering from Clostridoides difficile infection. The following are criteria for moderate to severe Clostridoides difficile infection:
[00266] I. Leukocytosis (white blood cell count >20x 109/L) 100267] 2. Plasma albumin level <30 ga, 1002681 3, Creatinine level >50% of baseline [002691 4. Hypotension (systolic blood pressure <100 mmHg) [002701 5. Fever (temperature >38 C) 100271] 6. Abdominal pain and distension [002721 7. Radiological evidence of colonic dilation, asches or ileus 1002731 1;Nample 4: Obesity 1002741 Obesity is associated with myriad sequelae including type II diabetes.
cardiovascular disease, some cancers, kidney disease, obstructive sleep apnea, gout, osteoarthritis, and many others. These frequently lead to a shortened lifespan. There is a strong positive correlation between weight loss and reduction of risk for these conditions.
Studies of fecal microbiota transplantation have shown that the procedure has the ability =
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 instigate obesity. This suggests that there is a micro biome component to obesity. Obesity is defined as a Body Mass Index (BMI) of >30 kg/m3. The objective of this example is to investigate the microbiome of obese individuals to examine the relative abundance of microbes contained therein.
1002751 The same procedure noted above for Example I was performed on 30 individuals suffering from obesity.
1002761 Example 5: Alzheimer's Disease 1002771 Alzheimer's disease (AD) is a neurodegenerative disorder and is the most common form of dementia. As of 2014 there were more than 5 million Americans living with Alzheimer's disease. The characteristic brain lesions, amyloid plaques and neurolibrillary tangles, cause progressive loss of cognitive function. The gut may play a major roll in this process. Dysbiosis of the gut microbiome can lead to systemic inflammation, which may in turn compromise the blood brain barrier, and lead to neuroinflammation and damage to neurons. The objective of this example to determine whether a specific microbe is present in individuals with Alzheimer's disease.
1002781 The same procedure noted above tbr Example I was performed on individuals suffering from Alzheimer's disease.
[00279] Example 6: Psoriasis 1002801 Psoriasis is a long-term skin autoimmune disease which causes patches of red, itchy, scaly skin. These patches can be small and localized or widespread.
Plaque Psoriasis is the most common type, accounting for 90% al cases. The most commonly affected areas are the forearms, skins, naval area, and scalp While it. is thought that genetics may play a role in the development ofPsoriasis, early sequencing studies of the gut rnicrobiome of Psoriasis patients have found the relative abundance of certain microbes to be altered in Psoriasis PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 patients. Thus. the balance of the microbiome may play an important role in Psoriasis development and treatment. The objective of this example to evaluate the similarities in the gut flora of different individuals with psoriasis and difference when compared to healthy individuals.
1002811 The same procedure noted above for Example 1 was performed on 30 individuals suffering from psoriasis.
1002821 Example 7: Autism 1002831 Autism spectrum disorders (ASD) are characterized by qualitative impairment in social interaction and communication skills, as well as stereotypic behaviors and limited activities and interests. As of 2014, 1 in 59 children in the United States will be diagnosed with ASD. In one sample set taken from several locations in the US, the rate of ASD
diagnosis went from I in 150 to 1 in 68 in just 10 years, more than doubling.
Core features of ASDs include verbal and nonverbal communication impairments, qualitative impairments in social interaction and the presence of maladaptive routines, repetitive behaviors and atypical interests or fixations. Comorbidity with at least one gastrointestinal symptom occurs in almost half of all children with ASD. The degree of severity of gastrointestinal symptoms strongly correlates to the degree of autism symptom severity. While some studies have identified specific microbes or families of microbes found to be perturbed in patients with ASD, evidence supporting positive impacts of altering the microbiome of individuals with ASD is in the very early stages. In one small study of oral vancomycin, short term improvement was seen with the majority of subjects, hinting at the strength of the gut-brain axis in the severity of ASD symptoms. The objective of this example is to evaluate the similarities in the gut flora of different individuals with autism and differences when compared to healthy individuals.

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Attorney Docket No. 20-42553 [002841 The same procedure noted above for Example I was performed on 30 individuals suffering from autism.
[002851 Exarnal.Q.8. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome 1002861 Chronic Fatigue Syndrome (CFS), also known as Myalgic Encephalomyelitis(ME) or ME/CFS, is a debilitating illness with no known cause, and no true treatment options. It also has no known cure. Patients with ME/CFS
experience profound exhaustion, unrefreshing sleep, joint aches and pains, post-exertional malaise, and frequently gastrointestinal problems. In a survey of drug use by ME/CFS patients there was found to be greater use of antacids, 112 blockers, and proton pump inhibitors than in the general population. Ba.cteriotherapy using oral and rectal probiotics has caused some improvement in patient's gastrointestinal symptoms. Thus dysbiosis is hypothesized to play a role in ME/CFS. The objective of this example is to evaluate the similarities in the gut flora of different individuals with Nclus and differences when compared to healthy individuals.
1002871 The same procedure noted above for Example 1 µN' as performed on 30 individuals suffering from ME/CFS.
1002881 Example 9: The Role of Diet [002891 The human gastrointestinal (GI) microbiome is a complex, interconnected 'web of microbes, living in a symbiotic relationship with their host. There are greater than ten times more bacteria in the human body than there are human cells, all in a delicate and ever-changing balance to maintain a healthy GI tract. When this balance is disrupted, a condition known as dysbiosis, disease can occur. There is still a debate over whether dysbiosis is a cause of disease or a symptom of it. Naturally, since the microbiome has such a profound impact on human health, including helping humans digest food, make vitamins, and teach their immune cells to recognize pathogens, there is a desire to study and learn as much about PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 the microbiome as possible. By correlating this data with survey data and medical records for the patients, connections may begin to be drawn between organisms present in the microbiome of the gastrointestinal tract, and disease. This is accomplished by comparing the answers of survey questions to disease states in participants. For example, if there is one particular microbe in patients with Crohn's disease, the data suggest that this microbe could play a role in the cause or progression of this disease. More importantly, only microbial activity within a family can be compared. The microbiotne is passed on from mother to child therefore it makes sense to compare microbiome of mother and child to understand better the microbiome Much like fingerprints, no microbiome is identical therefore, in order to understand a disease, it is preferred to look at the microbiome of a parent compared to a child or in an individual at baseline of healthy compared to a disease state. The objective of this example is to evaluate the similarities in the gut flora of different individuals with similar diet.
[00290] The same procedure noted above for Example I was performed on 30 individuals with similar diet, [00291] FAample JO COVID-I9 Infection [00292] COVID-19 is caused by a novel betacoronavirus (SARS-CoV-2) that is thought to have originated in bats in the city of Wuhan, China This disease has rapidly spread to become a worldwide pandemic. Scientists have identified the molecular structure of the spike glycoproteins on the surface olthe virus, which are what allow the virus to "stick" toils target, in this case the human lung The virus has a very similar sequence and structure to the SAM coronavinises, with the exception ofthe receptor binding domain Within a specific loop domain of the binding pocket of SARS-CoV-2, there is a change which replaces two proline residues with two flexible glycine residues, converting a rigid structure to something much more flexible, which is thought to facilitate stronger binding to the human host cell PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 ACE2 receptor, The ACE2 receptor is present in the lungs, however, it is also present in the intestine, kidneys, and testis. Thus, there is concern that the intestines could be a reservoir for the virus, and that the virus could be transmitted by the fecal oral route, in addition to transmission by aerosols It is critically important that patient stools be tested to determine if this is happening.
100293] There are many diseases for which the degree of dvsbiosis is a marker for disease severity. It is highly likely this phenomenon will also exist in the case of COVID-19. Thus, comparison between patients with different levels of severity will allow determination of whether it occurs with COVID-I9. The objective of this example to determine whether the virus is shed la the stool following negative RT-PCR testing and to correlate the microbiome sequencing data with information provided by patients and their medical records regarding COVID-1 9.
(002941 The procedure for this example is as Ibllows. The first step was collection of a COVID-19 sample. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected according to CDC protocol. Synthetic fiber swabs with plastic shafts were used. NP swabs were collected by insertion of a swab into the patient's nostril parallel to the palate. The swab is tell in place a few seconds to allow it to absorb secretions. OP swabs were collected by inserting the swab into the mouth without touching the tongue, cheek, or uvula. The tip of the swab was touched to the area around the tonsils and twisted five times to collect sufficient secretions for tinting 1002951 Following a positiNe test by RT-PCR, and again following subsequent negative test, patient stool samples were collected via the procedures noted above (stool sample collection kit or colonoscopy). Following fecal collection, individual patient DNA and RNA
was extracted and purified. The isolated DNA was quantitated utilizing a fluorometer. and the RNA was quantitated with a RNA quantitation system.

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Attorney Docket No, 2042553 1002961 ARer DNA quantification, the DNA was normalized and libraries were prepared utilizing shotgun methodology. This process utilized the shotgun workflow wherein samples undergo tagmentation, amplification and indexing, and purification.
1002971 After RNA quantification, the RNA was normalized and library fabrication was executed. This workflow included RNA fragmentation, first and second strand cDNA
synthesis, adenylation, adapter ligation, and amplification.
[002981 Samples libraries were normalized to create a library pool which is quantified and appropriately diluted to the final loading concentration to be sequenced on the appropriate sequencing system/machine 100299] Following completion of the NextSeq run, the raw.bc1 data was streamed in real time for conversion to FASTQ files. The FASTQ files were then pushed through the bioinformatics metagenomics pipeline with patient specific endpoint readouts praline each individual's unique microbiome.
100300] More specifically, the bioinformatics pipeline utilized computational tools that profiled the microbial communities from metagenomic sequencing data with species level resolution. Patient microbiome profiles were analyzed to ascertain not only the profile of microbes in patient samples but also to identify specific strains, and provide accurate estimation of orgainsmal abundance relative to the overall diversity.
1003011 Patient specific microbiome profiles were aligned to their medical records and other patient provided information for further analysis and interpretation.
1003021 The stool samples were retained for future use in a 20T
freezer.
[00303] Figure 30 is a flow chart of the method of sequencing the microbiome of an individual recovering from COVID-19 infection. The method comprises the basic steps of providing an individual that had been infected with CONTI D-19 300; providing a stool sample PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 from the individual 302; analyzing the microbiome of the individual 304, and freezing the stool sample from the individual for future use 306.
[00304] Eximigit. Role of Gut Flora in Disease 1003051 The objective of this example is to investigate the microbiome of individuals suffering from the following diseases or health conditions: C. difficile infection, Obesity, Autism, Alzheimer's disease, Crohn's disease, Myalgic Encephalomyelitis/Chronic, Fatigue Syndrome (ME/CFS), Psoriasis, Chronic UTI, Ulcerative Colitis, Multiple Sclerosis (MS).
Chronic constipation, Celiac sprue, Lyme disease, Elevated cholesterol, Colorectal cancer, Amyotrophic lateral sclerosis (ALS), Rheumatoid arthritis, Parkinson's disease, Depression, Anxiety, Obsessive-Compulsk e disorder, Bipolar Disorder, Migraine headaches, Diabetes mellitus, Lupus, Epidermolysis, /vletastatic mesothelioma, irritable bowl syndrome (IBS) Diarrhea, IBS Constipation, Eczema, Acne, Fatty liver, Myasthenia gravis, Gout.
[003061 The same procedure noted above for Example I was performed on at least 100 individuals suffering from each disease or health condition listed above.
[003071 Example 12: SARS-CoV-2 100308] Objective: SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. The objective of this example is to identify SARS-CoV-2 by enrichment NGS from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COV1D- 19 patients.
1003091 Methods: 14 study participants (n = 14) underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS. Following fecal collection, RNA
was extracted, reverse transcribed, and the library was prepped, enriched, and sequenced.
Sequences were then mapped to the S,ARS-CoV-2 Wuhan-Hu-1 (MN90847.3) complete genorne utilizing One Codex's SARS-CoV-2 bioinformatics analysis pipeline.
SARS-CoV-2 PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 2(1-42553 positive samples were further analyzed for mutational variants that differed from the reference genome. Of the 14 study participants, 12 also had their nasopharyngeal swabs tested for SARS-CoV-2 by RT-PCR.
1003101 Results: Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n 14). and RT-PCR nasopharyngeal swab analysis (n 12). The concordance of SARS-CoV-2 detection by enrichment NGS
from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS.
1003111 More specifically, the results from patients that had their stool samples tested by whole genome enrichment NGS were evaluated, as yell as their nasopharyngeal swabs were tested by RT-PCR for the presence of SARS-CoV-2. Of the 14 study participants, ten were symptomatic and tested positive for SARS-CoV-2 by RT-PCR, two asymptomatic individuals tested negative, and two other asymptornatic individuals did not undergo RT-PCR
testing (Table 24). Patients S and 7, that had tested positiµe by RT-PCR from nasopharyngeal swabs, were treated with Hydroxychloroquine (11CC)õAzithromycin, vitamin C, vitamin D, and zinc for 10 days prior to fecal collection. Similarly, after positive nasopharyngeal swab.
patient 13 was treated with vitamin C, vitamin 0, and zinc for 10 days before fecal collection.
The concordance of SARS-CoV-2 detection by enrichment NGS from stools among positive non-treated patients tested by RT-PCR nasopharyngeal analysis was 100% (7/7).
Patient 8, who did not undergo nasopharyngeal analysis, tested positive for SARS-CoV-2 by NOS. The three patients (5, 7. 13) that received treatment prior to providing fecal samples, all tasted negative by NGS. Asymptomatic patients 2 and 9, who tested negative by nasopharyngeal swab, were also negative by NGS, as NN as asymptomatic patient 14.
1003121 Table 24 documents the symptoms and SARS-CoV-2 testing results.

. . .
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Attorney Docket No. 2(1-42553 Nasopliaryngeal Sample 11) Symptoms Swab (RT- Treated Fecal Patient fli(S) Location PCItt ¨ ¨
Patient 1 febrile, diarrhea, anosmia, 02 sat. <90% + RO + PA
Patient 3 febrile, diarrhea, 02 sta. <90% ___ + no + CA
Patient 4 febrile, chairhea, anosmia, 02 sat. <90% + . no + AZ
Patient 6 febrile, cough, anosmla + no + AZ
Patient 8 none n/a 00 + CA
Patient 10 febrile, cough, headache + BO + GA
-----------------_____ Patient Ii febrile, cough, headache + no + GA
Patient 12 febrile, cough + no + GA
¨
Patient 5 febrile, cough + yes ¨ CA
_ Patient 7 febrile, cough .1. yes ¨ GA
Patient 13 febrile, cough + yes ¨ GA
Patient 2 none ¨ no ¨ CA
Patient 9 none ¨ , no ¨ CA
Patient 14 none Wit no ¨ CA
_ ______________________ Table 24 [00313] All fecal samples analyzed by enrichment NOS from positive patients by RT-PCR achieved 100% genome coverage or SARS-CoV-2 except for patient 3 which had 45%, and patient 10 which had 93% coverage (Table 25). The total number of SARS-CoV-mapped reads for patients 1, 3, 4, 6, 8, 10, II, and 12 were 465645, 5984, 131582, 793603, 496852, 5929, 1270734, and 38256 respectively. The mean read depths of SARS-CoV-2 for patients 1, 3, 4, 6, 8, 10, 11, and 12 's N'ere 1129.8x, 31.7x. 318.6x, 1924.6x, 1206.7x, 15 5x, 3075.3x, and 92.7x respectively. The read depths at specific coordinates along the SARS-CoV-2 genome for each patient are captured in Figure 31.
(00314] Table 25 documents the enrichment NOS metrics.
Sample ID Genome Number of Mapped Mean Coverage 'Variants Reads Depth Patient 1 100% 11 465645 1129.8x .
Patient 3 45% 11 5984 ___ 31.7x Patient 4 100% 9 131582 , 318.6x Patient 6 100% 10 793603 1924.6x Patient 8 100% 10 496852 1206 7 Patient 10 93% 9 5929 15.6x _ s . .
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Attorney Docket No. 20-42553 Number Genome f Mapped o MUM
Sampie ID
Coverage Reads Depth Variants Patient 11 100% 10 1270734 3075.3x ¨
Patient 12 100% 10 38256 92 7x Table 25 1003151 Following alignment and mapping of SARS-CoV-2, patient genomes were compared to the Wuhan-IN-I (MN90847.3) SARS-CoV-2 reference genome via One Codex's bioinformatics pipeline to identify mutational variations. This analysis identified nucleotide variants at positions n1241 (C --. T) and m23403 (A ---i. G) across all positive patients, and variants at positions nt3037 (C ----* T) and n125563 (0 --, T) in seven of the eight patients (Table 3) Interestingly, patients 8, I I, and 12 harbored the same set of variants, as did patients 4 and 6 (who were kindreds). Unique variants not identified in any of tbe other individuals were detected in patients I. 3, 6, and 10, with patient 3 harboring the most distinct SARS-CoV-2 genome with eight unique variants, followed hy patient 1 with seven.
Collectively, there were thirty-three different mutations among the patients in which SARS-CoV-2 was detected by whole genome enrichment NOS.
I00316] Table 26 documents the SARS-CoV-2 genomie positions, variant changes, and frequencies across the positive patient cohort.
Patient Patient Patient Patient Patient Patient Patient Patient Region (ORF) =Position Variant 1 3 4 6 8 '.. tan 2.# j C ---. T 100% 100% 100% 100%
100% 100% 100% 100%
. .
la 833 T --, C x X A X 100% X
100% 100%
1.1 1059 C ¨ T A A 100% t00%
99% 100% 100% 100%
It 1758 , C --, T X X 100% 100% x x x x la 1973 C ¨. T x x x 87% X X
X X
la 3037 C ¨ T 100% x 100% 100%
100% 100% 100% 100%
la 3078 C ¨+ T x 89% x , x , x x x x it 4g6t, G ¨ T , 75% x x x X . X , A
X
la 6720 C' ¨ T 93% X A A N N
X X
it KM , 0 --4 T x 100% x X X X
X X
1,1 _ 9401 _ T --t C x x x A A
04% X X

6 . .
PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 1 _____________________________ Patient Patient Patient Patient Patient Patient Patient Patent Region (ORP) p 00 ion Valiant i 1 3 4 6 8 10 11

12 , la 9403 T -4 A x X N 1 X
64% X X
---- , la 10870 G-.1 x x 1003 100% X
N N X
la 11123 Ci --. A x x 100% 100% x x X N
lb , 24408 C' ¨T 100% x 100% 100%
100% x 100"/n 100%
lb 14877 . C --' T x 100% , x x x x , x x lb 16616 C ¨ T x x x x 100% x 100% 100%
lb 16848 , C --IT 1001 . x x x x x x x lb 18652 C ¨ A x x x x a 83% _ x x lb 19989 T ¨. 0 x 100% N N X X
X X
Spike 2157ti I --. (3 x 83% x x x x x x ..... SP1/4......._ h...23264...._02.
A... _... x___ ....15%._. _ x _ A ---. A ................x ........... x......_...... x .......
Spike 23403 A --= G 100% 100% 1000/u 100% 100% 100% 100% 100%
Spike 23603 C ¨ T 82% x x x x x , x x 3a 25563 CT ¨ T x 100% 100% 100%
100% 10(PAt 100% 100%
3a 25970 C¨" A x x x x 100% x 100% 100%
8 27964 C ¨ T x x X X 100% X
100% 100%
¨ ¨
Nucleoprotein 28881 0 --. A 100% x x x x x X X
Nucleoprotein 28882 0 --, A 100% x x x x x x x - .
Nucleoprotein 28883 0 --= C 100% x x N X X
X X
Nucleoprotein 28997 C ¨T x 100%
Nucleoprotein 29019 A ¨ T x 100% µ x X X
X X
-Nucleoprotein 29364 C ¨ G x X N N x 85% x x Table 26 1003171 Discussion. Coronaviridae is a family of enveloped, single-stranded, positive-sense RNA viruses. The total length ofthe genome is 30 Kb, consisting of a 5'-termintil noncoding region. an open reading frame (ORE) I a/b-coding region, an S region encoding the spike glycoprotein (S protein), an E region encoding the envelope protein (E protein), an M region encoding the membrane protein (NI protein), an N region encoding the nucleocapsid protein (N protein), and a -3'-terminal noncoding region Among them, the poly protein encoded in the ORFI alb region of the nonstructural protein can be cut by 3CLpro and PLpro of the virus to (Orin RNA-dependent RNA polymernse and helicme, which guides the replication., transcription, and translation of the virus genome The NI and E
proteins are involved in the formation of the envelope, while the N protein is involved in assembly. The PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 spike protein binds to the receptor of the host cell and confers specificity for viral invasion into susceptible cells, [003181 It is believed this is the first study to report whole genome sequencing (WGS) of SARS-CoV-2 from stool samples. The study was able to identify SARS-CoV-2 in patients that tested positive by nasopharyngeal swab RT-PCR analysis and observed unique genomes in 62.5% of the NGS positive patients. The overall homology among the genomes was high (99,97%), with variations identified in the ORF regions la, lb, S, 3a, 8, and N. Of particular interest, was the adenine to guanine change in the S protein at position nt23403 which converts aspartic acid to glycine (D G).
The conversions of glycine to arginine (n128883) and proline to arginine (nt29.364) in the nucleoprotein are also of particular interest. While enrichment NGS is both costly and time consuming. these striking results highlight the potential viability of SARS-CoV-2 in feces, its possible role in transmission, and may accurately document complete eradication of the virus.
1003191 Fitufe __ 31 iff Figures 31A-31H are a series of graphs depicting whole genome alignment of SARS-CoV-2 in patients (Pt). The x-axis depicts the genomic coordinates as aligned to the NIN908947.3 reference genome, and the y-axis represents the read depth at specific loci. Figure 31A is patient 1.Eggre. 318 is patient 3. Figure 31C is patient 4. Figure 31D is patient 6, Figure 31E is patisnt 8 Figure 31F is patient 10. Figure 31G
is patient 11.
Figure 31H is patient 12.
1003201 Conclusion: These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal-oral transmission.
This study also elucidates the advantages of SARS-CoV-2 enrichment NOS, which may be a key methodology to document complete viral eradication PCT/us20/44605 26 May 2021 (26.05.2021) PCT APPLICATION
Attorney Docket No. 20-42553 [003211 Having thus described the invention, it should be apparent that numerous structural modifications and adaptations may be resorted to without departing from the scope and fair meaning of the instant invention as set forth herein above and described herein below by the claims.

Claims

What is claimed is:
1 A rnethod of testing for specific organisms in an individual, the method comprising the steps of a) screening the individual, b) acquiring a stool sample from the individual.
c) processing the stool sample to obtain the individuaLs microbiome, wherein the step of processing comprises the sub-steps of i) extracting DNA from the stool sample, \ hich comprises adding the stool sample to a bead beating tube, achieving cell 1 sis. capturing the DNA on a silica membrane in a spm-colunm, and washing and eluting the DNA from the membrane.
and purifying the extracted DNA, d) sequencing the nuerobiome of the individual. and e) analyzing the microbiome of the indn idual to determine v\ hether one or more specific organisms are present in the incln idual. whereby a health condition of the individual is determined.
The method of claim 1, wherein step b) comprises providing the individual \ith a stool sample collection kit 3 The method of claim 2. wherein the stool sample collection kit comprises.
a) at least one stool sample collection \ al, b) at least one toilet accessory, c) at. least one specimen bag.

d) at least one pair of gloves, e) an authorization form:
t) a patient inforrnation card.
g) a questionnaire; and h) stool sample collection instructions.
4 The method of claim 1, wherein step b) comprises acquiring the stool sample from the individual via colonoscopy 'The method of claim 1, wherein the one or rnore specific organisms of step e) comprise one or more of-the Colloss mg Acmetobacter baumannu. Actmomv ces odontolyticusõAkkermansia muciniphila, Bacillus cereus. Bacillus subtilis, Bacteriodes Bacteroides yulgatus, Bifidobacterium adolescent, Blastoostis hommis. Butym ibrio proteoclasticus. Campylobacter jejuni, Candida albicans. Chlamvdophda pneumoniae.
Clostridioides difficile. Clostridium berjermckii, Clostridium perfringens, Clostridium sporgesse, Crptococcus neoformans. Cunbacterium acnes. Demococcus radiodurans, Enterobacter cloacae, Enterococcus faecalis. Escherichia coli, Fusobacterium nucleatum, Helicobacter hepaticus. Helicobacter Klebsiella pneumomae, Lactobacillus gasseri.
Lactobacillus fermentum. Lactobacillus plantarum, Listeria monocv togenes. Ntv cobacterium av ium subsp paratuberculosis. Neisseria meningitides, Porphvromonas Proteus Pseudomonas aerugmosa. Rhodobactet sphaeroides, Saccharomyces cereµ isiae.
Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalachae, Streptococcus mutano. Streptococcus pneumomae, Streptococcus ogenes.
ToNtoplasma gondn. Yersima enterocolitica, and Bacteria X
6 The method of claim 5. wherein the organisms are selected from the group cornprising Acinetobacter baumannii, Actinornyces odontolvticus, Bacillus cereus, Bacillus Listeria monocytogenes, Proteus rmrabilis Pseudonionas aeruginosa.
Streptococcus agalactiae, Streptococcus mutano, Streptococcus prieumonme, and Streptococcus pyogenes.
7. The method of clairn 5, wherein the organisms are selected from the group comprising: Bifidobactermm adolescentis. Blastoc sUs hominis, Butyriyibrio proteoclasticus.
Clostridium betjerinckii, Clostridium perfringens. Clostridium sporgesse, Lactobacillus gassert, Lactobacillus fermentum. Lactobacillus plantarum. and Staphylococcus epidermidis 8. The method of claim 5, wherein the organisrns are selected from the group comprising. Carnpylobacter jejuni, Candida albicans, Chkunydophila pneumomae, Crptococcus neoformans, Cuubactenum acnes. Deinococcus radiodurans, Flelicobacter hepaticus, Porphyrornonas gingivalis. Rhodobacter sphaeroides. Saccharomyces cerevisiae.
Toxoplasma gondii, and Bacteria X
9 The method of claim 5. wherein the organisms are selected from the group comprising. Akkermansia muciniphila, Bacteriodes fragilis, Bacteroides vulgatus.
Clostridioides dilci1e. Enterobacter cloacae. Enterococcus faecahs, Eschenchia coh, Fusobacterium nucleatum, Helicobacter pylori, Klebsiella pneumomae, Mycobacterium aviurn subsp. paratuberculosis. Neisseria meningitides, Salmonella enterica, Staphylococcus aureus, and Yersmia enterocolitica 10. The method of claim 1, wherein step e) ls an assay that tests for the following organisms: Acinetobacter baumannu, Actinornyces odontolyticus. Akkermansia mucimpfula.
Bacillus cereus, Bacillus subtilis. Bacteriodes fiagilis. Bacteroides vulgatus, Bilidobacterium adolescent. Blastocystis hornims, Butyrivibrio proteoclasticus. Campylobacter jejuni, Candida albicans, Chlarnydophila pneumoniae. Clostridioides difficile, Clostridium beijerinckii. Clostridium perfrmgens. Clostridium sporgesse. Crptococcus neoformans, Cutibacterium acnes, Deinococcus radiodurans, Enterobacter cloacae, Enterococcus faecalis, Escherichia coh. F usobactenum nucleatum, Helicobacter hepaticus, Helicobacter Klebsiella pneumoniae, Lactobacillus gasseri, Lactobacillus ferrnentum.
Lactobacillus plantarum, Listeria monocµtogenes, Mycobacterium avium subsp paratuberculosis.
Neisseria meningitides, Porphyromonas gingivalis. Proteus rnirahilis, Pseudomonas aeruginosa, Rhodobacter sphaeroides. Saccharomyces cerevisiae, Salmonella enterica.
Staphylococcus aureus, Staphylococcus epidermidis. Streptococcus agalactiae. Streptococcus mutano, Streptococcus pneumonlae, Streptococcus pyogenes. Toxoplasrna gondn, Yersinia enterocohtica, and Bacteria X
11 The rnethod of claim 1, wherein step e) comprises comparing the microbiome of the individual to a trucrohionie Of a mother of the individual 12. The method of claim 1. wherein step e) comprises comparing the microbiome of the individual to a microbiome or a sibling of the individual 13 The method of claim 1. wherein step e) comprises comparing the microbiome of the individual with a health condition to a microbiome of another indiµ idual IN
ith the same health condition 14 The method of claim 1. wherein step e) comprises comparing the microbiome of the indiµ idual with a health condition to a microbiome of the indiµ idual before the individual had the health condition.
15 The method of claim 1, further comprising step t) after step et. storing the processed stool sample in a freezer 16 A method or determining whether an indi idual has a health condition, the method comprising the steps of a) acquiring a stool sample frorn the indn b) processing the stool sample to obtain the individual's microbiome, c) sequencing the microbiorne of the individual. and d) analyzing the microbiome of the individual to determine whether one or rnore specific organisms are present in the individual. whereby the health condition of the individual is determined.
17 The rnethod of clairn 16. wherein the health condition is selected from the group cornprising C. difficile infection, Obesity. Autism, Alzheimer's disease.
Crohn's disease, Myalgic Encephalomyelitis/Chronic, Fatigue Syndrorne (ME'CFS), Psoriasis.
Chronic Urinary' tract infection, Ulcerative Colitis, Multiple Sclerosis, Chronic constipation, (;eliac sprue, Lyrne disease, Elevated cholesterol. (;olorectal cancer. Amyotrophic lateral sclerosis.
Rheumatoid arthritis, Parkinson's disease, Depression. Anxiety, Obsessive-Compulsive disorder, Bipolar Disorder, Migraine headaches, Diabetes mellitus, Lupus, Epidermolysis, Metastatic mesotheliorna, irritable boyyel syndrome Diarrhea. irritable boyy el syndrorne Constipation, Eczema. Acne, Fatty/ lrvèr. Mvasthenia gravis, and Clout 18 The method of claim 16. Y.\ herein step b) comprises the steps of i) extracting DNA from the sarnple, which cornprises the steps of adding the stool sample to a bead beating tube, achieving cell lysis, capturing DNA on a silica membrane in a spin-column, and washing and eluting the captured DNA from the membrane, and ii) purifying the extracted DNA.
19 The rnethod of claim 16, wherein the one or more specific organisms of step are selected from the group consisting of Acmetobacter baumanniiõ,kchnomyces odontolyficus.
Akkermansia mucimphila, Bacillus cereus, Bacillus subfilis. Bacteriodes Bacteroides vulgalus, Bifidobacternim adolescent, Blastocvstis hommis, Butyrivibrio proteoclasticus.
Campylobacter jejum. Candida albicans. Chlamy dophila pneumonme.
Clostridioides Clostridium beijerinckii. Clostridium perfringens. Clostridium sporgesse.
Crptococcus neoformans, Cuubacterium acnes, Demococcus radiodurans, Enterobacter cloacae, Enterococcus faecalis, Escherichia coh. Fusobacterium nucleaturn, Helicobacter hepaticus, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus gassen, Lactobacillus fermentum, Lactobacillus plantarum, Listeria monocytogenes, Mycobacterium aµ !um subsp.
paratuberculosis. Neisserta meningitides, Porphyromonas gingivalis. Proteus Pseudomonas aeruginosa. Rhodobacter sphaeroides. Saccharom ces cereµ isiae, Salinonella enterica. Staphylococcus aureus, Staph. lococcus epidernadis. Streptococcus agalactme, Streptococcus mutano, Streptococcus pneumoniae. Streptococcus pyogenes, Toxoplasma gondh, Yersinia enterocohtica. and Bacteria X
")() A stool sample collection kit comprising=
a) at least one stool sample collection vial.
b) at least one toilet accessory.
c) at least one specimen bag.
d) at least one pair of Lloves:
e) an authorization form.
f) a patient information card.
g) a questionnaire. and h) stool sample collection instructions