Note: Descriptions are shown in the official language in which they were submitted.
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PCT/US2020/044605
1
METHOD OF TESTING FOR SPECIFIC
ORGANISMS IN AN INDIVIDUAL
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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
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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
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(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
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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,
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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,
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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:
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[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;
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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.
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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
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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
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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:
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[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.
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[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:
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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
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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:
4
-
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.
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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.
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[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.
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[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
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[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
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[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.
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[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
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[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;
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[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.
1002061 EXAMPLES
[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
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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
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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
1 1 1 1 1 1 1 1 19
bollette ___________________
JClostridiuM) 1 1 u 1 1 1 1 0 1 1 1
1 1 1 1 1 1 1 1 17
seindens
[Clostridium] 1 1 1 1111 t 1
I I I 19
saccharolvt icun
[Clostridium] 1 1 1 I 1 1 1 0 1 1 1
1 1 1 1 1 1 I 1 18
.s2henoides
[Clostridium] 1 1 1 1 1 1 0 1 1 I 1
11 1 1 1 I 1 18
Clostridiales i 0 1 I 1 1101
11111111 1 17
baclenum
CCNA10
Closiridiales 1 0 1 I I I I 0 I 1 1
1 1 I 1 1 1 I1 171
bacterium
70B-A
Clostridium 1 1 1 1 1 1 1 0 1 1 1
1 1 1 1 1 1 1 18
sporogenes
Clostridium 1 1 0 1 1 1 1 0 1 1 1
1 1 1 1 1 1 1 1 17
sp. SY8519
Clostridium 0 1 1 1 0 1 1 1 1 1 1
1 1 1 1 1 1 1 17
botulinum 202F
Clostridium 1 1 1 1 1 1 1 0 1 1 1 0 1
1 1 I III 1 17
botulinum 13
Clostridium 0 U 0 0 0 0 1 () 1 1 1 1 1
1 1 1 I 1 1 17
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
117
bolulintint B1
;-
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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
1 1 1 1 1 I 0 I 17
botulinum
H04402
Clostridium 0 1 1 1 I 0 0 0 1 I 0
1 1 0 0 1 II 0 II
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
1 1 1 0 1 1 1 0 14
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
1 1 1 1 1 1 1 1 17
3_
Clostridium I 0 -7 1 1 1 1 0 1 1 1
1 1 1 1 1 1 1 1 11-1
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
1 1 1 I 17
baratii
Clostridi tun 1 1 1 1 1 1 1 0 1 I 1
1 1 I I 1 1 1 1 18
sp. CT4
Clostridium 1 1 1 1 I I 1 0 I 1 I
1 1 1 1 1 11 1 18
pastourianum
BC I
Clostridium 1 0 0 1 0 0 1 0 1 1 1
I 1 1 1 1 I 1 1 14
burmii sir.
Clostridium 1 1 1 1 I 1 1 0 1 1 1
1 1 1 1 1 I I 18
isatidis
Clostridium 1 0 1 0 1 1 1 0 1 171 I
1 1 I 1 16
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
1 1 1 1 I 1 I 18
acetiCUM
Clostridium 1 1 1 1 I 1 1 1 1 1 1 1
1 1 1 1 I 1 1 14
septictun
Clostriditun 1 0 0 1 0 1 1 0 1 I 1 1
1 1 1 I 1 1 1 15
itoµyi NT
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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
1 1 1 I1 1 18
I
argentinense . .
C lostri di um 1 1 1 I 1 I 0 0 1 1 1 1
1 1 I 1 1 1 1 17
bomimense
Clostridium 1 I 1 1 1 1 1 1 1 I 1 1 I
1 1 1 1 1 1 19
sp. BNL1100
Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1
1 1 1 1 1 1 18
cochlearium
Clostridiwn 1 1 1 1 1 1 1 0 1 1 1 1 1
1 1 1 1 1 1 18
sp. JN50090 I _
, _ . . ,
,
Clostridium 1 1 1 1 1 1 1 0 I 1 1 I 1
I 1 1 I 1 1 18
sp. IN-9
Clostridium 1 I 0 I 1 1 0 (I I 1 I 0 1
1 1 1 1 1 1 15
sp. JN-I . . _
Clostriditun 1 I 1 1 1 1 0 1 0 1 1 1 1
1 1 1 1 1 1 11
Saccharo=
. butylicum 1
,
, , , , , ,
Clostridium 1 1 1 1 1 1 1 0 1 1 1 I 1
1 1 1 1 1 1 18
, tyrobutyricum _
_ .
Clostridium 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 19
estertheticum ...... ,
Clostridium 1 1 1 1 1 ' 1 I -0 1 ' 1 1 0 1 1
' 1 - 1 1 I 1 17
Carboxidivorans
P7
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
1 1 1 1 1 1 1 18
chauwei
Clostridium I I) 0 1 I 1 1 1 1 1 0 1 1
1 0 I 1 1 1 15
Clostridium 1 1 1.1 I 1 1 1 1 I 1 1 I 1
1 1 1 1 1 19
tetani
Clostridiun) 1 I 1 1 1 1 1 0 1 1 1 1 1
1 0 1 1 1 1 17
!Wain
12124569
Clostridium 1 1 1 1 1 1 1 0 1 I 1 1 1
1 0 1 1 0 1 16
tenni E88
Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1
1 1 1 I 1 1 18
scatologenes ,
Clostridium 1 0 1 1 1 1 0 0 1 1 1 1 1
1 1 1 1 1 1 10
tueniosporum . . ¨ . .
Clostridium 1 I I 1 1 1 1 1 1 1 1 1 1
1 1 0 I 1 1 18
drake] I .
Clostridium 1 1 1 1 1 I 1 0 1 1 1 1 1
1 1 1 1 1 1 18
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
1 0 1 1 0 I 16
ljungdallIti
DSM
Clostridiaceae 1 I 1 1 1 1 1 0 0 1 1
1 1 1 1 1 1 1 1 17
bacterium
I4S0207 -
Clostridioides 1 1 1 1 1 1 1 1 I 1 1
1 1 I 1 1 1 1 1 19
dilEcile . _ _
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Org. Name Cont. A B C 1) E 1 2 3 4 ¨5 ¨6 7 8 9 10 11 12 13
14 T
Claitridioides 1 1 1-1 1 1 1 0 1 1 1 1
1 1 1 1 1 1 I18
difficile ATCC
C lost ridioldes 0 0 0 0 0 I 1 0 1 1 1
1 1 1 1 1 1 1 1 13
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
1 1 1 1 1 1 1 1 16
innocuum
'Clostridium' 1 0 1 0 1 1 1 0 0 1 1
I 1 1 1 1 1 1 1 15
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
CCINIA10
Closuidialos 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 3
bacterium
70B-A
[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
1 1 1 1 I 1 1 14
kluyvori
Clostridium 0 0 0 1 ¨0 1 1 1 1 0 0 0 1 0 0 0 1 0 7
collulovorans
743B
Clostridium
0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 1 1 1 10
Saccharoper-
bulylacetonicum
Total 59 59 57 60 64 65 56 26 70 75 69 70 70 1 70 59 70 76 66
66
Clostridia
Tecies
26 75 59
76 66 66
Table 4
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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
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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
140010059
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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
DSM 44634
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
1CM 15466
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).
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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
51449 1 1 1 1 4
7 7 7 7
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
7
Bacteroides I 7
Bacteroides fss 638R ____________________ 1 0 1 1 1 1 1
6
Bacteroides fragilis YCH46 1 0 1 1 1 I 1
6
Bacteroides fragilis NCTC 9343 1 0 1 1 1 1 1 6
Helicobacter hepaticus 1 0 1 0 1 1 1
5
Helicobacter he.pliticus ATCC 51449 I0 1 _!.L I 5
7 2 7 5 7 7 7
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
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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
6
Yersinia pestis Antigua () 0 I 0 , 0 0 0
1
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
7
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
7 -
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
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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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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
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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).
1002411
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
387 FT-00H
FT-0012 s Bacteroides fragilis
1,382,505 FT-0012
Table 15
1002421
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
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[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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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
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(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 ).
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[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.
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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
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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
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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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[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
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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
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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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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
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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
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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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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-
2
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
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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
10 11 12
'.. 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
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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
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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
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[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.
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