DESC:F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: GUT MICROBIAL BASED PROFILING FOR DISEASE SUSCEPTIBILITY ASSESSMENT AND PERSONALIZED NUTRITIONAL RECOMMENDATION

2. APPLICANT (A) NAME: LEUCINE RICH BIO PVT LTD

(B) ADDRESS: # 283, 3RD FLOOR, PUTTALINGIAH RD, GURURAJA LAYOUT, PADMANABHANAGAR, BENGALURU, KARNATAKA 560070

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
[001] TECHNICAL FIELD OF THE INVENTION
[002] The present invention is in the technical field of gut microbial based profiling for disease susceptibility assessment and personalized nutritional recommendation.
[003] BACKGROUND OF THE INVENTION
[004] Human gut harbours largest microbial community in human body and comprises somewhere between 300 and 1000 different microbial species.
[005] The human body consists of trillion cells, and the gut microbiome contains at least two orders of magnitude more genes than are found in human genome.
[006] So there is a need to dissect the functional interactions between microbial communities and their environmental or animal habitats in order to understand human wellness.
[007] ‘Microbiome’ refers to the entire habitat of the microorganisms (bacteria, archaea, lower eukaryotes, and viruses), their genomes, and the surrounding environmental conditions [Ex. human gut]1. This definition is based on that of biome, the biotic and abiotic factors of given environments. Further, the term ‘Microbiota’ is defined as the entire community of microorganisms present in a defined environment [such as human gut or skin], while the term ‘Metagenome’ is the collection of genomes and genes from the members of a microbiota1,2. In the recent past, the distinct physio-chemical properties, including but not limited to microbial structures, metabolites, mobile genetic elements of the microbiome, has been reported to play a significant role in human health and long-term wellbeing.
[008] Due to their symbiotic/parasitic nature, these microbes play a significant role in many fundamental life processes3. Its ability to establish an axis with several extraintestinal organs, such as kidneys, liver, cardiovascular, the bone system and the brain, has gained a lot of traction in the recent past4. It has also emerged as a virtual metabolic and endocrine organ, given its impact on a large spectrum of digestion and metabolism of the food that we consume5,6,7. The gut microbiome has also been shown to be linked with certain chronic diseases such as IBD, type 2 diabetes, non-alcoholic fatty acid liver disease (NAFLD)8–12 etc. Because of such significant impact, manipulation of the gut microbiome (composition, abundance, and diversity) has been deemed to significantly influence our physiological functions, through immune and metabolic regulation.
[009] Such manipulations are currently achieved through dietary changes, pre and probiotic supplementations13,14 [such as synbiotics, parabiotics, psychobiotics etc.], and recently Faecal Microbiota Transplantations (FMT)15–17 - for serious clinical conditions like Clostridium difficile infections and others. Recent reports have even suggested that these microbial communities within the gut, can modify (both potentiate and weaken) the drugs consumed to treat specific disease conditions18,19. This has started a dialogue on the need for in-depth knowledge of the impact of the microbiome on therapeutic strategies and have gained huge momentum with the precision medicine domain1,20.
[010] Majority of organisms have not been cultured in the laboratory, and furthermore, it is necessary to determine the effect of gut microbial community on a organism.
[011] So, there is a need to perform microbiome analysis in the field of microbiology, focused on the health and wellness domain of public health sector. In particular, the amount, identity, presence, and/or ratio of gut microflora (Bacteria, Archaea, Fungi, Virus, Metazoa and Protozoa) in a subject, to recommend one or more dietary and probiotic-centred approach to improve overall health and wellbeing.
[012] In summary, there is an urgent need to develop a product and a method for gut microbial based profiling for disease susceptibility assessment and personalized nutritional recommendation.
[013] SUMMARY OF THE INVENTION
[014] The primary objective of the present invention is to provide a gut microbial based profiling for disease susceptibility assessment and personalized nutritional recommendation. More particularly, invention deals with gut microbiome as a biomarker and/or therapeutic target for a condition in a subject. Furthermore, invention provides methods of assessing, altering, providing personalized nutritional recommendation and monitoring the condition in the subject. In addition, the invention also provides kits that are useful for the practice of the methods of the invention.
[015] According to the exemplary aspect of the present invention, the product BugSpeaks® - Characterize human gut microbiota profile (composition, abundance and diversity), establish current health status, and thereafter providing a comprehensive and customized evidence-based dietary, probiotic and prebiotic recommendation to improve and sustain overall health and wellness of an individual.
[016] In an embodiment, a method for predicting disease susceptibility by profiling microbiota, comprising the steps of: registration of user; dispatching the kit; obtaining sample comprising the gut microbiome from a human patient to be diagnosed; extracting DNA; sequencing DNA; analyzing the data; concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient; predicting disease susceptibility; and recommending a personalized nutritional regimen based on individual’s gut microbiota.
[017] According to a further aspect of the present invention, the invention is particularly relevant, in the fields of Microbiome, Gut Microbiome, Metagenomics, High-throughput sequencing technology, Precision medicine, Health and Wellness, Direct to Consumer (DTC) omics Alternative treatments, and Nutrition and diet.
[018] According to aspects of the invention provides, comprehensive analysis of Bacterial, Archaeal, Fungal, Viral, Metazoal and Protozoal composition and abundance of the gut microbiome (not limited to just Bacterial).
[019] Health and wellness, rather than clinical. This removes those clinical barricades and focus turns to DTC process/product.
[020] Since focus is health and wellness, the characterization of one's gut microbiome enable us to provide actionable recommendations, which will aid the individual in improving his/her overall health and wellbeing.
[021] The actionable well-tolerated recommendations, which is the key differentiator, include,
[022] Three Phase dietary recommendation - with specific list of foods and frequency tags (frequent, moderate, low and avoid).
[023] Probiotic recommendations - to rejuvenate the gut microbiome, to promote gut microbial composition, with lasting effects on overall health,.
[024] Prebiotic recommendations - to aid the rejuvenation process of the gut microbiome.
[025] These recommendations are strictly evidence based with reported associations with diseases, traits and overall health and wellbeing.
[026] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
[027] BRIEF DESCRIPTION OF THE DRAWINGS
[028] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[029] FIG. 1 illustrates the bird-eye view for the invention, the workflow and the process of Bug Speaks., according to the aspects of the invention.
[030] FIG. 2 illustrates the schematic representation of Methods of Holding Tube, according to the aspects of the invention.
[031] FIG. 3 illustrates the schematic representation of Ease of Opening the kit, according to the aspects of the invention.
[032] FIG. 4 illustrates the schematic representation of Inclined Position of the Tube, according to the aspects of the invention.
[033] FIG. 5 illustrates the schematic representation of Ease of handling the tube, according to the aspects of the invention.
[034] FIG. 6 illustrates the schematic representation of Inclination allowing package flip, according to the aspects of the invention.
[035] FIG. 7 illustrates the schematic representation of Accommodating Info pack, according to the aspects of the invention.
[036] FIG 8A illustrates the schematic representation of Concept and different designs of the Bugspeak Box, according to the aspects of the invention.
[037] FIG. 9 illustrates the schematic representation of Over the box grooving and design, according to the aspects of the invention.
[038] FIG. 10 shows the Finished product, according to the aspects of the invention.
[039] FIG. 11 shows the Stool Collection Tube, according to the aspects of the invention.
[040] FIG. 12 Illustrates a block diagram illustrating the details of a digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules.
[041] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[042] DETAILED DESCRIPTION OF THE INVENTION
[043] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[044] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[045] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[046] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[047] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[048] Example embodiments of the present invention are described with reference to the accompanying figures.
[049] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[050] EMBODIMENTS OF THE INVENTION:
[051] In an embodiment, a method for predicting disease susceptibility by profiling microbiota, comprising the steps of: registration of user; dispatching the kit; obtaining sample comprising the gut microbiome from a human patient to be diagnosed; extracting DNA; sequencing DNA; analyzing the data; concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient; predicting disease susceptibility; and recommending a personalized nutritional regimen based on individual’s gut microbiota.
[052] In further embodiment, the disease susceptibility is provided based on individual’s gut microbiota.
[053] In an embodiment, the said disease conditions are Irritable Bowel Syndrome, Colorectal Neoplasm, Constipation, Diabetes Mellitus Type 2, Inflammatory Bowel Disease, Non-Alcoholic Fatty Liver Disease, Ulcerative Colitis, Aerobic Endurance, Crohn’s Disease, Physical Endurance, Clostridium Difficile Infection, Power, Depression, Prone to Fatigue, Anxiety, Gluten intolerance, Rheumatoid Arthritis, Asthma, Chronic Kidney Disease, Eczema, Atherosclerosis, Atopic dermatitis, Obesity, Acne, Hypertension, Effect of Smoking, Parkinson’s Disease, Autism spectrum disorder, Alzheimer’s disease, Preeclampsia or any combination thereof.
[054] In an embodiment, a method for profiling microbiota, comprising the steps of: obtaining raw data; checking quality of data; mapping on Microbial Database (MetaRef); doing Taxonomical Profiling; estimating Abundance Estimation; plotting Generation; analysing diversity; mapping on MicroByte database; estimating or detecting Disease Risk; mapping on NutrBite database; and recommending a personalized nutritional requirement.
[055] In an embodiment, the method for analysing the microbiome comprises computational analysis on MetaRef, MicroByte, NutriBite and MetaRich data bases.
[056] In further embodiment, the method for estimating the disease risks is based on microbiome of the individual.
[057] In an embodiment, the said detecting comprises a method, such as a quantitative method, selected from the group of PCR, rtPCR, qPCR, multiplex PCR, high-throughput sequencing, metatranscriptomic sequencing, identification of strain-specific markers, such as genes and/or proteins, and 16S rDNA analysis.
[058] In an embodiment, a method for monitoring the progress of microbiome-based disease condition comprising the steps of: obtaining sample comprising the gut microbiome from a human patient to be diagnosed; extracting DNA; sequencing DNA; analyzing the data; concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient; monitoring the disease progress; and recommending a personalized nutritional requirement.
[059] A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method, and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
[060] In an embodiment, a kit comprising: kit contents and guide; a pair of hand gloves; stool collection frame; stool collection instructions; stool collection tube; protective bubble envelope with desiccant; questionnaire and informed consent; return envelope; and an instruction booklet.
[061] As highlighted above, the influence of microbiome can be exploited due to its malleability and can be personalized because of its uniqueness in every individual. However, personalizing the kind and quantity of the dietary, probiotic, and prebiotic recommendations, can only be achieved after characterizing one’s unique gut microbiome. Gut microbiota’s link to various diseases as highlighted above provides a window to address them and provide solutions. Hence, evaluating (composition, abundance, and diversity) the gut microbiome seems to be the most promising path forward, at the same time a key rate limiting stage for achieving optimal health benefits, and this invention resolves this exact problem at hand.
[062] BugSpeaks® is an innovation that has compiled a process for analysing the gut microbiome of an individual, providing an end-to-end DTC solution involving sample collection, processing, sequencing, analysing, characterizing the microbial composition (Bacteria, Archaea, Fungi, Virus, Metazoa and Protozoa) of the individual. Followed by establishment of associations between the gut microbiome profile, overall health status, and providing a comprehensive and customized list of dietary, probiotic, and prebiotic recommendations. Hence, BugSpeaks® can aid in making guided decisions about one’s lifestyle and the recommendations if followed to a recommended phased duration, can significantly improve the overall health and wellbeing of the individual. BugSpeaks® will provide important health information and actionable inputs to improve overall health of the individuals. Disease risk assessment based on the gut microbiota can provide the end user with the know-how to tackle the diseases in advance through dietary, lifestyle and medicines. This can improve the overall health ecosystem as well as many diseases can be nipped in the bud or such guidance will provide alternate/supplement solutions to deal with an individual’s health.
[063] Process of BUGSPEAKS
[064] User-> Registration->Questionnaire-> Kit dispatch-> Sample collection->Kit Return-> DNA Extraction-> DNA Sequencing->Analysis-> Reporting.
[065] Technical Process of BUGSPEAKS
[066] Raw Data-> Quality Check -> Microbial Database (MetaRef)-> Mapping->Taxonomical Profiling->Abundance Estimation->Plot Generation->Diversity Analysis->MicroByte database Mapping->Disease Risk Estimation->NutrBite database Mapping->3 Phase Nutritional Recommendation.
[067] Specifically, such guided decisions can be made in:
[068] Nutritional management (of pregnant women to neonates to adults to geriatric individuals). Dietary recommendations are largely beneficial, with no or minimal negative impact on our health.
[069] Various probiotic and prebiotic have been recognized as an effective alternative or at least supplement the conventional treatments and managements of various diseases (largely gastroenteric).
[070] Such probiotic and prebiotic interventions, along with already established dietary standards, can undoubtedly have a positive outcome on disease management and over health.
[071] Well tolerated set of dietary, probiotic, and prebiotic recommendations, for manipulation of one's gut microbiota is the next big thing in disease management and sick care.
[072] The goal is to manipulate and sustain [through diet or supplementation] a gut microbiota enriched with commensals that can maintain healthy physiological (metabolic, immune, hormonal, neural, behavioural, cognitive, and emotional) functions. This has also opened exciting prospects for exploiting the microbiota for personalized nutrition, medicine, and beyond.
[073] EXAMPLE EMBODIMENTS- 1
[074] The innovation is a process, and the process is for comprehensive characterization of the microbiome of an individual and provide health status of the individual highlighting possible risk to certain diseases based on the microbiota profile of the gut, in order to make guided decisions about one’s diet, supplementation and over lifestyle.
[075]
[076] The process comprises of:
[077] A system for collecting the stool sample [representing the microbiome] of an individual - Sample collection (Designed kit – Section-3A)
[078] A process for processing and sequencing the metagenome of the sample [representing the microbiome] of an individual - DNA Extraction and Sequencing (Section-3B)
[079] A method for analysing the microbiome of the individual, comprising - Computational Analysis (MetaRef, MicroByte, NutriBite & MetaRich – Section-3C)
[080] A method for estimating the disease risks based on microbiome of the individual - Evidence based interpretation (Disease Scoring Matrix – Section-3D)
[081] Providing dietary, prebiotic, and probiotic recommendations based on microbiome of the individual - 3 Phase Diet (Section-3E)
[082] A system for collecting the stool sample [representing the microbiome] of an individual Sample collection (Kit Design)
[083] SECTION-3A
[084] Inventors developed and compiled a process for analysing the gut microbiome of an individual, comprising an end to end DTC solution involving sample collection, processing, sequencing, analysing, characterizing the microbial composition (Bacteria, Archaea, Fungi, Virus, Metazoa and Protozoa) of the individual. Followed by establishment of associations between the gut microbiome profile and overall health status, and providing a comprehensive and customized list of dietary, probiotic and prebiotic recommendations, which if followed to a recommended duration and phase, significantly improve the overall health and wellbeing of the individual.
[085] As mentioned above, the innovation is a process and the Process includes the following key steps,
[086] Sample collection (Designed kit – Appendix 1),
[087] DNA Extraction,
[088] Sequencing,
[089] Computational Analysis (MetaRich, MetaRef, MicroByte & NutriBite - Appendix 2), and
[090] Evidence based interpretation (Disease Scoring Matrix, 3 Phase Diet – Appendix 3).
[091] The innovation (process) has been successfully used to process hundreds of stool samples of individuals, with seamless generation of dietary, probiotic and prebiotic recommendations. Inventors have exclusively tested the process and the finer aspects of it (viz., accuracy of disease risk estimations and other associations), in small study. The study included 15 patients with IBD, IBS, ODS (5 patients with each condition) collectively called as FGIDs or Functional gastrointestinal disorder and analyzed their stool for establishing overall gut microbiome profile, estimate risk of the disease and other downstream characterizations. The present invention was also tested in a small cohort of patients suffering with depression and atherosclerosis as proof of concept.
[092] BugSpeaks® is a registed Trademark of LEUCINE RICH BIO PVT LTD.
[093] To begin with the BugSpeaks(R) process was seamlessly implemented from sample collection to sequencing to MetaRich pipeline, which could analyze characterize sequencing data as unique gut microbiome profile. The key validation from this study was the algorithms ability to estimate the risk of the disease. The scoring algorithm (Appendix 3) could accurately estimate the risk and was correlated clinically with other parameters. Overall the study could thoroughly validate the process. Inventors are currently tracking susceptibility to the 30 diseases based on gut microflora profile as mentioned in the table below:-
[094] TABLE A: 30 diseases based on gut microflora profile
Irritable Bowel Syndrome Colorectal Neoplasm
Constipation Diabetes Mellitus Type 2
Inflammatory Bowel Disease Non-Alcoholic Fatty Liver Disease
Ulcerative Colitis Aerobic Endurance
Crohn’s Disease Physical Endurance
Clostridium Difficile Infection Power
Depression Prone to Fatigue
Anxiety Gluten intolerance
Rheumatoid Arthritis Asthma
Chronic Kidney Disease Eczema
Atherosclerosis Atopic dermatitis
Obesity Acne
Hypertension Effect of Smoking

Parkinson’s Disease Autism spectrum disorder
Alzheimer’s disease Preeclampsia
[095] Invention provides following benefits,
a. Comprehensive analysis of Bacterial, Archaeal, Fungal, Viral, Metazoal and Protozoal composition and abundance of the gut microbiome (not limited to just Bacterial)
b. Health and wellness, rather than clinical. This removes those clinical barricades and focus turns to DTC process/product
c. Since focus is health and wellness, the characterization of one's gut microbiome enable us to provide actionable recommendations, which will aid the individual in improving his/her overall health and wellbeing
d. The actionable well-tolerated recommendations, which is the key differentiator, include
i. 3 Phase dietary recommendation - with specific list of foods and frequency tags (frequent, moderate, low and avoid)
ii. Probiotic recommendations - to rejuvenate the gut microbiome, to promote gut microbial composition, with lasting effects on overall health
iii. Prebiotic recommendations - to aid the rejuvenation process of the gut microbiome
[096] The second key differentiator is that, these recommendations are strictly evidence based with reported associations with diseases, traits and overall health and wellbeing.
[097] The third key differentiator is the ability to predict susceptibility to certain diseases based on the gut microflora (Appendix 3). This unique proposition will aid in better lifestyle management including but not limited to customized food intake (recommendation) also provided by this unique offering and thus leading to healthy living.
[098] Similar processes that characterize intestinal bacterial diversity using similar analytical methods and new generation of high-throughput sequencing technology, does exist. Some examples given below. Problem's or Drawbacks of these include (but not limited to):
a. Largely involves 16s sequencing and bacterial / archaeal characterization only (while we encompass all)
b. Targeting therapeutic and diagnostic value of the analysis. Hence have a lot of clinical barricades
c. The solitary focus on either therapeutic or diagnostic value of it, limits itself to only achieving that (nothing beyond this).
d. Focused on characterizing microbiome associated with either treatment (say antibiotics) and / or associated with solitary disease conditions (such as asthma, allergy, obesity, metabolic syndrome etc.)
[099] Usually a method for making a synthetic microbial ensemble to improve mostly a solitary target biological property in a target biological environment.
[0100] EXAMPLE EMBODIMENTS- 2
[0101] WORKFLOW AND THE PROCESS OF BUG SPEAKS
[0102] Workflow is illustrated in FIG 1.
[0103] Kit Box and components of Kit Box:
[0104] FIG 2- FIG 11 shows different aspects of Kit Box.
[0105] Methods of Holding Tube
a. 9 Different methods were tested
b. Finalized on 1 - Figure A
c. A single neck holder allowing for flip (see below) (FIG 2)
[0106] Ease of Opening the kit
a. Ease of opening the kit box was tested with 12 different forms
b. Finalized on 1 - Figure I (FIG 3)
[0107] Inclined Position of the Tube
a. Inclined Position of the tube should allow for ease of taking out from the kit
b. 8 Different methods were tested
c. Finalized on 1 - Figure C (FIG 4)
[0108] Ease of handling the tube
a. Inclined Position of the tube should allow for ease of taking out from the kit
b. 6 Different methods were tested
c. Finalized on 1 - Figure A (FIG 5)
[0109] Inclination allowing package flip
a. The inclination should allow for logistical trauma, like flipping and tossing
b. The tube must remain in an inclined position allowing for the sample to be always immersed within the protective solution
c. 3 different designs with different angles of inclination were tried
d. Finalized on 1 - Figure A (FIG 6)
[0110] Accommodating Info pack
a. The designed kit should allow for placement and secure shipment of information pack - which includes a questionnaire for analysis, consent form to sign, and instructions manual.
b. The information packet should be secure from any spillage from the tube.
c. 8 different concepts were tried - Finalized on 1 - Figure D (FIG 7).
[0111] Concept
a. After implementation of all the above final concept was tested as 3 different forms
b. Finalized on 1 - Figure A (FIG 8)
[0112] Over the box grooving and design
a. The BugSpeaks Logo placement and other final touches on the kit was finalized as shown (FIG 9)
[0113] Finished product
a. The final product with all the finalized design aspects looks like this, along with BugSpeaks branding. (FIG 10)
[0114] Kit Contents:
[0115] To collect stool:
[0116] Kit contents and guide
[0117] A pair of hand gloves
[0118] Stool collection frame (for commode)
[0119] Stool collection instructions
[0120] Stool collection tube (with sample stabilizing solution)
[0121] To return after stool collection:
[0122] Protective bubble envelope with desiccant (to place the collection tube)
[0123] Questionnaire and informed consent (Filled & signed by the user or attending)
[0124] Return Envelope (to return the stool sample for processing)
[0125] Instruction Booklet
[0126] An instruction booklet is provided to inform the user on how to collect the stool sample. The booklet also gives instruction to the user on the procedure to send the sample back to the LRB.
[0127] Stool Collection Tube:
[0128] The tube consists of a transparent cylindrical tube with a screw top. The screw top has a spatula attached to it. The spatula has been designed to collect 1mg of the stool. The tube contains a liquid stabilization buffer. The DNA Stabilizer effects inactivation of DNases and prevents degradation of DNA, it preserves the microorganism titre and pre-lyses bacteria. The stabilized sample can be stored in the Stool Collection Tube during 3 months at room temperature. (FIG 11)
[0129] Stool Collector Frame
[0130] The stool collector paper comprises of a simple paper loop which is slipped over the toilet seat to collect the sample. The paper has been designed to be strong enough to hold the stool, while a small test sample can be taken. Following this, the paper can be torn at each side and allowed to fall into the toilet bowl and can be flushed away. The paper has been designed to be completely degradable.
[0131] Gloves: A pair of plastic gloves are provided to facilitate clean collection of the stool sample.
[0132] Questionnaire: A detailed questionnaire is provided. The questionnaire contains questions related to the following categories:
[0133] Name, Age, Sex and Email Id
[0134] Food Habits
[0135] Disease Conditions, if any.
[0136] Return Envelope:
[0137] To place the sample, filled in questionnaire and any other forms. Protective bubble envelope [with desiccant to absorb any spillage] to place the collection tube with sample. Return envelope with inbuilt adhesive sticker to securely pack and ship.
[0138] A process for processing and sequencing the metagenome of the sample [representing the microbiome] of an individual
[0139] SECTION-3B
[0140] DNA Extraction and Sequencing
[0141] DNA will be extracted from stool samples received at the processing facility using fast purification protocol of genomic DNA.
[0142] Later, whole genome shot gun sequencing will be performed on samples using short read sequencing. Raw sequencing reads will be stored in FastQ format for further computational analysis.
[0143] A method for analysing the microbiome of the individual, comprising:
[0144] Computational Analysis
[0145] HARDWARE
[0146] Digital Processing System
[0147] Fig 12 is a block diagram illustrating the details of a digital processing system in which various aspects of the present invention are operative by execution of appropriate execution modules, firmware or hardware components. Digital processing system 500 may correspond to any of monitoring devices 120a-120c or server system 150.
[0148] Digital processing system 500 may contain one or more processors (such as a central processing unit (CPU) 501), random access memory (RAM) 502, secondary memory 503, graphics controller 506, display unit 507, network interface 508, and input interface 509. All the components except display unit 507 may communicate with each other over communication path 505 which may contain several buses as is well known in the relevant arts. The components of FIG. 9 are described below in further detail.
[0149] CPU 501 may execute instructions stored in RAM 502 to provide several features of the present invention. CPU 501 may contain multiple processing units, with each processing unit potentially being designed for a specific task. Alternatively, CPU 501 may contain only a single general-purpose processing unit. RAM 502 may receive instructions from secondary memory 503 using communication path 505.
[0150] Graphics controller 506 generates display signals (e.g., in RGB format) to display unit 507 based on data/instructions received from CPU 501. Display unit 507 contains a display screen to display the images defined by the display signals. Input interface 509 may correspond to a keyboard and a pointing device (e.g., touch-pad, mouse), which enable the various inputs to be provided.
[0151] Network interface 508 provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface 508 may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of WIFI, Bluetooth based communication).
[0152] Secondary memory 503 may contain hard drive 503a, flash memory 503b, and removable storage drive 503c. Secondary memory 503 may store the data and, which enable digital processing system 500 to provide several features in accordance with the present invention.
[0153] Some or all of the data and instructions may be provided on removable storage unit 504, and the data and instructions may be read and provided by removable storage drive 503c to CPU 501. Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are examples of such removable storage drive 503c.
[0154] Removable storage unit 64 may be implemented using storage format compatible with removable storage drive 503c such that removable storage drive 63c can read the data and instructions. Thus, removable storage unit 504 includes a computer readable storage medium having stored therein computer software (in the form of execution modules) and/or data.
[0155] However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). These “computer program products” are means for providing execution modules to digital processing system 500. CPU 501 may retrieve the software instructions (forming the execution modules) and execute the instructions to provide various features of the present invention described above.
[0156] Digital processing system may correspond to each of user system: local system or remote and server noted above. Digital processing system may contain one or more processors (such as a central processing unit (CPU)), random access memory (RAM), secondary memory, graphics controller (GPU), primary display unit, network interfaces like (LAN, Wi-Fi), and input interfaces (not shown).
[0157] CPU executes instructions stored in RAM to provide several features of the present invention. CPU may contain multiple processing units, with each processing unit potentially being designed for a specific task. Alternatively, CPU may contain only a single general purpose processing unit. RAM may receive instructions from secondary/system memory.
[0158] Graphics controller (GPU) generates display signals (e.g., in RGB format) to primary display unit based on data/instructions received from CPU. Primary display unit contains a display screen (e.g. monitor, touchscreen enabled monitor) to display the images defined by the display signals. Input interfaces may correspond to a keyboard, a pointing device (e.g., touch-pad, mouse), a touchscreen, etc. which enable the various inputs to be provided. Network interface provides connectivity to a network (e.g., using Internet Protocol), and may be used to communicate with other connected systems. Network interface may provide such connectivity over a wire (in the case of TCP/IP based communication) or wirelessly (in the case of Wi-Fi, Bluetooth based communication).
[0159] Secondary memory may contain hard drive (mass storage), flash memory, and removable storage drive. Secondary memory may store the data (e.g., the specific requests sent, the responses received, etc.) and executable modules, which enable the digital processing system to provide several features in accordance with the present invention.
[0160] Some or all of the data and instructions may be provided on a removable storage unit (SD card), and the data and instructions may be read and provided by removable storage drive to CPU. Floppy drive, magnetic tape drive, CD-ROM drive, DVD Drive, Flash memory, removable memory chip (PCMCIA Card, EPROM) are other examples of such removable storage drive.
[0161] Software Architecture:
[0162] Removable storage unit may be implemented using storage format compatible with removable storage drive such that removable storage drive can read the data and instructions. Thus, removable storage unit includes a computer readable storage medium having stored therein executable modules and/or data. However, the computer (or machine, in general) readable storage medium can be in other forms (e.g., non-removable, random access, etc.). CPU may retrieve the executable modules and execute them to provide various features of the present invention described above.
[0163] SECTION-3C - MetaRef, MicroByte, NutriBite and MetaRich.
[0164] APPENDIX – 2 - MetaRich, MetaRef, MicroByte and NutriBite
[0165] Microbial Genome Database (MetaRef)
[0166] Largely, most of the open source tools for analysis of metagenomic data uses NCBI (National Centre for Biotechnology Information) database for creating their databases. However, NCBI's database does not offer complete and non-redundant coverage of genome data.
a. The RefSeq Genomes database is a comprehensive collection of NCBI Reference Sequence Genomes across all taxonomic groups that has more complete coverage and less redundant data than chromosome.
b. The RefSeq Representative Genomes database contains the best quality genomes (Reference and Representative Genomes) available at NCBI and provides broad taxonomic coverage with minimum redundancy.
[0167] Hence, inventors have chosen NCBI's RefSeq project (RefSeq Genomes database and RefSeq Representative Genomes database) for building our Microbial Genome Database.
[0168] Any degree of redundancy that still exists was corrected employing the following filtering criteria, while retaining our objective of creating a comprehensive database, preferably at strain level resolution (subject to availability).
[0169] a. Genomes belonging to all six kingdoms of microbes, viz, Bacteria, Archaea, Fungi, Protozoa, Virus and Metazoa, were downloaded from NCBI's RefSeq project (RefSeq Genomes database and RefSeq Representative Genomes database).
[0170] b. First, Representative/Reference genomes were chosen (if available), which represents the highest quality genomes.
[0171] c. If there was no "Representative/Reference genomes" available for a given organism, inventors picked the genomes based on following assembly levels, in order of preference viz., Complete Genome > Chromosome > Scaffold > Contig.
[0172] d. Any redundancy at assembly levels were further filtered out using Genome Length (higher length was preferred), Assembly quality (N50 value) and
[0173] The most important differentiator here is that the process has been completed automated and can be used to update our database consistently at regular intervals. Inventors are currently performing upgrades of our database every 3 months.
[0174] MicroByte – Microbiome Database
[0175] A comprehensive database of manually curated, evidence-based associations between human microbiota and diseases/traits.
[0176] The database is being compiled using a blend of 'auto-curation' through Natural Language Processing (NLP) and in-house custom algorithms, followed by a stage of rigorous manual validation.
[0177] The auto-curation sieves through published evidences, extracting a combination of disease and microbial associations, along with possible mechanisms of disease pathogenesis and/or traits.
[0178] Bio-medical publications related to case-control studies of the human gut microbiome are also tabulated in this database.
[0179] While auto-curation significantly reduces the time and efforts, the manual validation ensures establishing evidence-based associations between microbiota (composition and abundance) and diseases / traits.
[0180] The database MicroByte as a whole, a comprehensive collection of such evidence-based associations, enabling us to utilize this data at disposal, and in combination with MetaRich and NutriBite.
[0181] NutriBite – Nutrition Database
[0182] An in-house, in silico validated database integrating current knowledge of gut microbiome, microbial metabolism, disease and nutrition (and in extension foods).
[0183] The database is an effort to interlink and integrate these resources to establish nutritional associations between gut microbiome, its metabolic pathways and processes to diseases and traits, with the overall objective of formulating a set of dietary recommendations.
[0184] Inventors have made extensive efforts to capture and network microbiota, microbial genes, metabolic reactions, metabolites, nutrients, foods and human diseases, to reconstruct the associations between human microbiota (composition and abundance) and diseases/trait, with food (nutrients) as a critical function.
[0185] Inventors achieve this through creation and utilization of two major components of this database,
a. Food database
i. Contains list of all food (Indian and Western foods), encompassing ~1000 different foods and 15 food categories.
ii. Along with this, the database would also contain the sub-nutrients (belonging to major nutrient categories like carbohydrates, fats, vitamins etc.) which are important entrants to the metabolic pathways.
b. Microbial pathway database
i. Since most of gastroenteric activity, like digestion, absorption and metabolism, is carried out by microbial communities in gut, it was critical to compile all microorganisms, with metabolic annotation and pathway information.
ii. Contains gene orthologous data of ~5200 microorganisms, corresponding reactions details, including but not limited to, IDs of organic compounds, reactions they are involved in etc.
[0186] Inventors are also trying to achieve the critical inclusion of both microbial and human (host) metabolic pathways, since these nutrients and sub-nutrients from foods are usually metabolized by both host and the microbiota.
[0187] With these compiled data, inventors can integrate the nutrient and sub-nutrient information with that of human and host metabolic pathways, to devise dietary recommendations.
[0188] Hence, the critical utility of NutriBite largely resides in the devising a customizable dietary recommendation regime (since the microbial composition of one’s gut is unique), that assists an individual in coping with currently predisposed diseases, significantly improve his/her health and work towards establishing a balanced gut health and overall wellness.
[0189] The database is updated every week.
[0190] Probiotic Database
[0191] As many as 150 probiotics species were identified and compiled from several publicly available databases, which were deemed to have GRAS (Generally Recognized As Safe) Status and were studied for their beneficial properties.
[0192] To build the database of probiotics,
a. We made the probiotic species was assigned the GRAS status
b. We curated for natural sources
c. We curated for commercially available probiotic products, including these species
[0193] After these automated and manual curation stages, 46 of 150 probiotic species were deemed to meet our criteria and were included in the database.
[0194] Among these, 18 species were curated to have natural sources and 19 species were available as commercial products in India
[0195] The database is updated every month.
[0196] MetaRich – Analysis Pipeline
[0197] Leveraging the expanding prospects of microbiome research, inventors have integrated a robust computational pipeline "MetaRich" into a user-friendly cloud-based platform for comprehensive analyses of any microbiome data. After rigorous testing & validation of several leading microbiome analysis pipelines, tools & databases, inventors have compiled “MetaRich” to analyse both 16s rDNA & shotgun metagenomes, duly supplemented by our in-house computational tools. MetaRich will assist the BugSpeaks® process of discovering biological insights within complex microbial systems, ease the arduous task of large-scale microbiome data analysis and general actionable recommendations.
[0165][0164] Data Quality:
[0166][0165] The process checks for the data quality for any errors, such as incorrectly assigned base, low quality confidence of base call, over-represented sequences, percentage of Ns. etc., that might have gotten introduced during sequencing/basecalling. The collated quality statistics of the FastQ data would allow inventors to identify any errors and remove any low quality sequencing data, ensuring only quality passed data go into further analysis.
[0172][0166] One such quality measure is the "Per-Base sequence quality", which is measured using a Phred Score (Quality score) that tells us how many of the bases assigned are of low quality and needs to be trimmed out. It gives us the mean, median statistical significance test values to infer the quality of the sequence. If a sequencer is unable to make a base call with enough confidence, then it will normally substitute an N rather than a conventional base call this plots out the percentage of base calls at each position for which an N was called. Any such low Phred Score bases/reads would be removed in next steps.
[0173][0167] Data Quality Trimming:
1. After the FASTQ data is checked for quality, the first step is to remove low-quality base calls [Phred score of <30] and trim off from the 3' end of the reads.
2. Adapter sequences will also be removed from the 3’ end of reads. Brielfy, the algorithm analyses the first 1 million sequences of the FASTQ file and attempts to find the first 12 or 13bp of the adapters, allowing inventors to remove them from the data.
3. Only those sequences that pass all such thresholds will be deemed suitable for analysis and would proceed to next step.
[0174][0168] Data Processing:
1. Firstly, the host genome sequences (Homo sapiens) are removed from quality processed FastQ data, by aligning the reads in the FASTQ file to the reference human genome GRCh38. The unmapped reads are taken for further analysis ensuring only microbial sequences are used for microbial identification and characterization.
2. All unmapped reads are aligned with the microbial genome database (MicroByte), indexed as 19 genome index files. The alignment process would generate sequence alignment temporary files (SAM files), which are merged to create a single SAM file for further processing.
3. Some improvements measures will be taken to increase the number of reads correctly getting assigned to the microbial genomes, along with reduce the number of false positive genomes getting identified. Further, this module estimates proportions of reads from each genome by reassigning ambiguous reads. The algorithm performs a realignment process based on Bayesian strategy and an expectation maximization algorithm, where a value is assigned based on the reference genome length and the alignment score. Based on the alignment score, penalties are assigned to each ambiguous reads. Finally, the reads with unique alignments are retained and use them to guide the reassignment of reads with ambiguous alignments, and remove any ambiguous alignments thereafter.
4. Based on the alignment, a biom table (Biological Observation Matrix) is generated with the micro-organism taxonomic ids assigned from the microbial genome database. Species level taxonomic classification (NCBI taxonomic classification) is assigned for each Taxon ID. This biom table is the core data type for downstream analysis. It is a matrix of read counts of observations on a per-sample basis. Most commonly, the observations are OTUs (Operational Taxonomic Unit) or taxa, where one organism is be considered as one OTU.
5. The biom table is filtered based on a least abundant cutoff of 5 reads and other quality measure to improve the data further.
6. Lastly, the reads are converted to the relative abundance by calculating the percentage of reads assigned to each organism as compared to the total raw abundance of all microbial genomes. A level wise (phylum to species) categorization of these matrices are created with percentage normalised abundance.
[0198] Data Representation:
1. From the biom table a taxonomic tree in the form of a cladogram is created, which represents abundance of respective organism. Bar graphs of abundance at each level is created.
2. Alpha diversity (or within-sample diversity), which describe the richness and/or evenness of taxa in a single sample are calculated from biom table and represented in the form of boxplot.
[0199] A method for estimating the disease risks based on microbiome of the individual
[0200] SECTION–3D
[0201] Evidence based interpretation (Disease Scoring Matrix)
[0202] APPENDIX – 3
[0203] Evidence based interpretation (Disease Scoring Matrix, 3 Phase Diet)
[0204] Scoring and Risk Estimation
1. With the current BugSpeaks test inventors are performing the risk estimation for a total of 30 diseases, largely covering the prominent FGIDs, considering the direct correlations between gut microbiota.
2. All these associations are exclusively extracted from out inhouse database MicroByte (described above) and manually validated to be associated with 30 diseases.
[0205] Overall, the following key stages is followed during disease risk estimation:
[0206] While testing a given BugSpeaks sample, the sequencing data from the sample is first be passed via our analysis pipeline MetaRich, which begins with preprocessing and quality improvement measures.
[0207] Once done, all the microorganisms present within the sample will be identified by sequence similarity to that of our reference database MetaRef.
[0208] Once identified, the relative abundances and its % normalized abundances of these microorganisms within the samples is estimated.
[0209] The relative % abundances of 3 taxonomic levels (Family, Genus and Species) is then extracted for the sample.
[0210] Later, each microorganism is tagged, either as Decreased or Increased, depending on its relative abundance with respect to other microorganisms within the sample.
[0211] Once this sample specific tagging was completed for the test sample, these tags were then matched with the master list of risk associations (compiled from MicroByte Database), to create matrices for each of the 30 diseases (Table A).
[0212] Each disease matrix is then passed through an ingenious decision matrix inbuilt into the scoring system, which would assign risk/safe tags for each of the microorganisms associated with that specific disease.
[0213] Finally, inventors estimate the cumulative risk association for a specific disease, using a statistical measure that collectively takes each risk/safe tags assigned to each microorganism associated with that diseases
[0214] This process is repeated for all 30 diseases/ traits, to generate an output of all risk estimates to be reported.
[0215] Probiotic Recommendations
[0216] For a given test sample, after the relative abundance were estimated, all identified microorganisms were scanned for presence of probiotic species.
[0217] List of all identified microorganisms were matched with the probiotics database and all the probiotic species present in the test sample were retrieved.
[0218] Further, based on the relative % abundances (at species level) of these identified probiotic species, they were tagged as
[0219] High – if the relative % abundance within the sample was > 5%
[0220] Low – if the relative % abundance within the sample was >0% and < 5%
[0221] Absent – if the probiotic species was not identified within the test sample.
[0222] These tags represented the abundance of these probiotic species within a gut sample.
[0223] Furthermore, based on these % abundances inventors could also provide probiotic recommendations, as
[0224] Maintain – if it was already high in abundance in your gut
[0225] Increase – if in low abundance or absent in your gut
[0226] Along with this, the natural sources of these probiotics and the availability of commercial products containing these probiotic species were also provided, retrieved from our Probiotics Database.
[0227] Providing dietary, prebiotic, and probiotic recommendations based on microbiome of the individual
[0228] SECTION-3E
[0229] 3 Phase Diet, Prebiotic and Probiotic Recommendations
[0230] Dietary Recommendations
[0231] To devise customized dietary recommendations for any given test sample, inventors extensively utilized the NutriBite database (described above), which contains curated food database and microbial pathway information.
[0232] Briefly, inventors utilize NutriBite to integrate the food and microbiome pathway information with the unique set of microorganisms identified within the test sample and its relative abundance.
[0233] Through this integration inventors devise customized dietary recommendations for each sample tested that can be implemented into a diet plan by the individual.
[0234] The process begins with the raw abundance file, containing all the identified organisms within the sample, its Taxon IDs and relative % abundances.
[0235] The matched organisms, between the test sample and the microbial pathways, would now contain reaction details, with substrates and products.
[0236] At this point inventors utilize the food database of NutriBite, to integrate nutrient and sub-nutrient data with that of the reactions retrieved in the previous step.
[0237] Any food, with one of its sub-nutrients acting as a substrate for one or more of the microbial pathways, inventors pick that food for further validations.
[0238] Once this integration is complete for all sub-nutrients of all foods, inventors ideally get a large list of foods and associated microorganisms that can potentially metabolize and utilize these foods.
[0239] Further, these compiled food lists were distributed into 3 phases and each having one of the four frequency tags sets of dietary recommendations.
[0240] Our approach to restore the gut balance is based on a three-phase strategy:
[0241] Phase 1
[0242] Restoring your gut microbiome - 2 Weeks
[0243] Involves restoration or resetting of your gut microbiome, where inventors minimize the composition and abundance of pathogenic or opportunistic microorganisms, to create a gut environment ideal for beneficial microorganisms to grow in Phase 2. This phase requires strict changes in your diet for a short period of time and supplementation with anti-inflammatory foods, natural antibiotics, and through restriction of selected inflammatory foods.
[0244] Phase 2
[0245] Rebuilding your microbiome - 8 Weeks
[0246] Involves rebuilding of your healthy gut microbiome, through re-inoculation and replacement with mostly beneficial microorganisms. Inventors achieve this through incorporation of prebiotics and probiotics, via natural dietary sources and commercially available supplements. This lasts for up to 10th week of your diet plan (a total of 8 weeks), which ensure the complete restoration of your gut microbiota.
[0247] Phase 3
[0248] Maintaining the healthy gut - For at least 3 Months
[0249] Largely involves a streamlined method for sustaining the healthy gut microbiome built during phase 2. These dietary, prebiotic and probiotic recommendations can be adopted for long term sustenance, spanning up to 3 months of your diet plan.
[0250] All 3 phases have a total of 6 food categories, each containing a list of foods and a frequency tag. Inventors have used a total of 4 frequency tags that indicates how frequently you can include a specific food in your meal plan.
[0251] Frequent - can be consumed twice in a day [in 2 meals/day]
[0252] Moderate - can be consumed once in a day [in 1 meal/day]
[0253] Low - can be consumed once in 2 days [every alternate day, in 1 meal/day]
[0254] Avoid - Avoid the consumption as much as possible
[0255] Inventors devise the phases and the frequencies, based on
[0256] The foods ability to contribute to the overall gut health
[0257] Which further is dependent on the composition and relative abundance of microorganisms in the sample.
[0258] A food was deemed to be “included” in the diet, if the relative abundance of beneficial microorganisms utilizing this food is more than the abundance of pathogenic/non-beneficial microorganisms utilizing the same food
[0259] Similarly, a food was deemed to be “excluded” from the diet, if the relative abundance of beneficial microorganisms utilizing this food is less than the abundance of pathogenic/non-beneficial microorganisms utilizing the same food.
[0260] The key point here is, since these microorganisms and their relative abundance are unique to the individual being tested, the food list compiled would in extension be personalized.
[0261] These customized dietary recommendations were further fine-tuned, by reviewing the published evidences of these foods being reported as good or bad for overall gut health.
[0262] Appendix – 4: Complete flow of the BugSpeaks® Process
[0263] Registration
[0264] Payment
[0265] Fill up of Questionnaire for Analysis
[0266] Sample collection kit dispatch (Logistics)
[0267] Sample collection
[0268] Return of sample collected (Logistics)
[0269] DNA Extraction
[0270] Whole DNA extraction: DNA Extraction
[0271] In the first steps of the protocol, stool samples are lysed Proteins are digested and degraded under denaturing conditions incubation with proteinase K. Purified, concentrated DNA is eluted from the spin column in a low-salt buffer equilibrated to temperature (15–25°C).
[0272] DNA Sequencing
[0259] Genomic DNA is isolated from the sample and fragmented physically or chemically, leaving random 5’ and 3’ end overhangs. The resulting DNA fragments are then purified for the desired size (150bp), using magnetic beads. Following that, the end overhangs created from fragmentation are repaired into blunt ends, achieved using a combination of a 3’ to 5’ exonuclease and a 5’ to 3’ polymerase. The 3’ ends of the fragments are additionally adenylated; this single base overhang hybridizes with the 3’ thymine overhang of the adapters which are then ligated together. This collection of adapter-ligated fragments forms the library, which is then sequenced in a next generation sequencing machine. The output of the sequencing, a FASTQ file, is used in the next step.
[0273] Whole genome shotgun (WGS sequencing)
[0274] Predefined protocol
[0275] MetaRich
[0276] Pre-processing
[0277] Reporting
a. Gut Microbiome Index (GMI)
i. Composition
ii. Abundance
iii. Diversity
iv. GMI Score
b. Disease Risk Estimation
c. 26 diseases / traits Disease Risk Estimation
i. 30 diseases / traits
ii. 25 diseases [mostly FGIDs]
iii. 5 - lifestyle traits / diseases
d. 3-phase customized diet
i. Dietary recommendations
ii. Probiotics recommendations
iii. Prebiotics recommendations
[0278] Reporting and follow up.
[0279] Report Dispatch
[0280] Appendix – 5: Disease risk estimation process
1. Sequencing data from sample
2. MetaRich Analysis Pipeline
a. Pre-processing and quality improvement measures
b. Match sequences to reference database MetaRef
c. Identify all microorganisms within the sample
d. Estimate relative abundance of each microorganism
e. Estimate normalized % abundance microorganism
3. Extract relative % abundances of 3 taxonomic levels (Family, Genus and Species)
4. Tagged, either as Decreased or Increased (with respect to other microorganisms)
5. Matched with the master list of risk associations (compiled from MicroByte Database)
6. Create matrices for each of the 30 diseases
7. Passed through an ingenious decision matrix
8. Assign risk/safe tags for each of the microorganisms associated with that specific disease
9. Cumulative risk association for a specific disease
[0281] EXAMPLE EMBODIMENTS- 3
[0282] The innovation (process) has been successfully utilized to characterize microbiomes of hundreds of individuals, with seamless generation of customized dietary, prebiotic, and probiotic recommendations. We have exclusively tested the process and its finer aspects with data generated from these microbiomes, especially pertaining to the sensitivity and specificity of risk estimations of diseases/traits.
[0283] To begin with, the MetaRich analysis could characterize the sequencing data into unique gut microbiome profiles of individuals. The key validation from these experimental data confirmed our algorithm’s ability to estimate the risk of diseases/traits we screen for, using the MicroByte database [containing the associations between these diseases/traits with that of the microbiome]. The scoring algorithm [Discussed in Section-3D] could accurately estimate the risk of specific diseases and was correlated with clinical diagnosis of the individuals [as consented and disclosed by them].
[0284] As described under Section-3D, the cumulative risk association for a specific disease will be estimated after assigning risk/safe tags for each of the microorganisms associated with that specific disease [based in MicroByte], overlayed with abundance data of the individual. Later, the score was estimated on a scale of 0-100, and were further categorized into the following three thresholds:
[0285] Low-Risk [With risk scores >0 and <40]
[0286] Moderate-Risk [With risk scores >=41 to >=70]
[0287] High-Risk [With risk scores >=71 to 100]
[0288] While these thresholds of risk assessment were fixed for qualitative reporting, a comprehensive sensitivity and specificity assessment was performed for all diseases, with a cutoff value set at >=40.
[0289] Although BugSpeaks® screens for a total of 30 diseases/traits, the experimental data provided below pertains to 3 specific diseases, representing its corresponding broader class. We have provided the sensitivity and specificity data of our risk estimation algorithms for:
[0290] Inflammatory Bowel Disease (IBD) – representing the ability to estimate the risk of most FGIDs [Ex. IBS, Constipation etc.]
[0291] Atherosclerosis – representing the ability to estimate the risk of other cardiovascular diseases.
[0292] Depression - representing the ability to estimate the risk of other mental or lifestyle disorders.
[0293] Inflammatory Bowel Disease (IBD)
[0294] There is now ample evidence to show that gut microbiota has a major role to play in IBD21–25. The current methods of diagnosis are expensive, mostly invasive, and have lower diagnostic accuracy. Lab [blood or stool] tests check for anemia or infection, or the recent rise of Faecal Calprotectin as a diagnostic tool for IBD, all look for signs of inflammation at large, which is a broader marker of IBD26–28. Further, most of the current diagnostic methods, including endoscopic procedures, work well only when combined with alarming symptoms29,30.
[0295] The BugSpeaks® cumulative risk scoring, estimated utilizing the evidence-based risk/safe tags for many microorganisms associated with IBD [from MicroByte] and its abundance [from Individual], has a continuous scale of 0-100. To estimate the sensitivity and specificity statistics, we utilized 40 sample data, of which 20 were diagnosed with IBD [as consented and disclosed by individuals] and 20 were Non-IBD controls. All data were received from patients with their informed consent.
Sample ID Diagnosed Risk Call Risk Score TP/FN
Sample_1 IBD Moderate 45 True Positive
Sample_2 IBD Moderate 42 True Positive
Sample_3 IBD Moderate 41 True Positive
Sample_4 IBD Moderate 42 True Positive
Sample_5 IBD Moderate 55 True Positive
Sample_6 IBD Moderate 61 True Positive
Sample_7 IBD Moderate 48 True Positive
Sample_8 IBD Moderate 48 True Positive
Sample_9 IBD Low 35 False Negative
Sample_10 IBD High 74 True Positive
Sample_11 IBD High 73 True Positive
Sample_12 IBD High 71 True Positive
Sample_13 IBD Moderate 54 True Positive
Sample_14 IBD Moderate 63 True Positive
Sample_15 IBD Moderate 47 True Positive
Sample_16 IBD Moderate 49 True Positive
Sample_17 IBD Moderate 46 True Positive
Sample_18 IBD Moderate 44 True Positive
Sample_19 IBD Moderate 43 True Positive
Sample_20 IBD Moderate 50 True Positive
Table 1 – De-identified samples of patients with IBD and Bugspeaks disease susceptibility call

[0296] Sensitivity =

Total Samples
20
True Positive False Negative
19 1
Sensitivity
0.95

Sample ID Diagnosed Risk Call Risk Score TN/FP
Sample_21 Non-IBD Low 37 True Negative
Sample_22 Non-IBD Low 39 True Negative
Sample_23 Non-IBD Low 32 True Negative
Sample_24 Non-IBD Low 33 True Negative
Sample_25 Non-IBD Low 24 True Negative
Sample_26 Non-IBD Low 39 True Negative Total Samples
Sample_27 Non-IBD Low 31 True Negative 20
Sample_28 Non-IBD Low 32 True Negative True Negative False Positive
Sample_29 Non-IBD Low 33 True Negative 17 3
Sample_30 Non-IBD Low 39 True Negative Specificity
Sample_31 Non-IBD Low 25 True Negative
Sample_32 Non-IBD Low 31 True Negative
Sample_33 Non-IBD Low 30 True Negative
Sample_34 Non-IBD Low 40 True Negative
Sample_35 Non-IBD Low 33 True Negative
Sample_36 Non-IBD Low 28 True Negative
Sample_37 Non-IBD Low 33 True Negative
Sample_38 Non-IBD Moderate 41 False Positive
Sample_39 Non-IBD Moderate 42 False Positive
Sample_40 Non-IBD Moderate 41 False Positive
Table 2 – De-identified samples of control patients without IBD and Bugspeaks disease susceptibility call

Specificity =

[0297] Overall, based on the cutoff score of 40 or greater, we estimated the sensitivity of risk estimation of IBD by BugSpeaks® to be 0.95 [19/20 tests] and the specificity of the disease to be 0.85 [17/20 controls].
[0298] Atherosclerosis
[0299] Although there are few tests that can predict the occurrence of Atherosclerosis, like high cholesterol, high blood pressure etc., confirmation is required by angiography to locate the extent of damage, which is expensive and invasive. Recently microbial factors have gained traction as early indictors of atherosclerosis31,32–35.
[0300] The BugSpeaks® cumulative risk scoring, estimated utilizing the evidence-based risk/safe tags for many microorganisms associated with Atherosclerosis [from MicroByte] and its abundance [from Individual], has a continuous scale of 0-100. To estimate the sensitivity and specificity statistics, we utilized 20 sample data, of which 10 were diagnosed with Atherosclerosis [as consented and disclosed by individuals] and 10 were Non-Atherosclerosis controls. All data were received from patients with their informed consent.
Sample ID
Diagnosed Risk Call Risk Score TP/FN
Sample_1 Atherosclerosis Low 39 False Negative
Sample_2 Atherosclerosis Moderate 45 True Positive
Sample_3 Atherosclerosis Moderate 54 True Positive
Sample_4 Atherosclerosis Moderate 50 True Positive
Sample_5 Atherosclerosis Moderate 54 True Positive
Sample_6 Atherosclerosis Moderate 59 True Positive
Sample_7 Atherosclerosis Moderate 41 True Positive
Sample_8 Atherosclerosis Moderate 44 True Positive
Sample_9 Atherosclerosis Moderate 41 True Positive
Sample_10 Atherosclerosis Moderate 43 True Positive
Total Samples
20
True Positive False Negative
9 1
Sensitivity
Table 3 – De-identified samples of patients with atherosclerosis and Bugspeaks disease susceptibility call.
Sample ID Diagnosed Risk Call Risk Score TN/FP
Sample_1 Non-Atherosclerosis Low 39 True Negative
Sample_2 Non-Atherosclerosis Low 38 True Negative Total Samples
Sample_3 Non-Atherosclerosis Low 39 True Negative 20
Sample_4 Non-Atherosclerosis Low 38 True Negative True Negative False Positive
Sample_5 Non-Atherosclerosis Low 39 True Negative 7 3
Sample_6 Non-Atherosclerosis Moderate 40 False Positive Specificity

Sample_7 Non-Atherosclerosis Moderate 45 False Positive
Sample_8 Non-Atherosclerosis Low 39 True Negative
Sample_9 Non-Atherosclerosis Moderate 43 False Positive
Sample_10 Non-Atherosclerosis Low 36 True Negative

Table 4 – De-identified samples of control patients without atherosclerosis and Bugspeaks disease susceptibility call.
Overall, based on the cutoff score of 40 or greater, we estimated the sensitivity of risk estimation of Atherosclerosis by BugSpeaks® to be 0.90 [9/10 tests] and the specificity of the disease to be 0.70 [7/10 controls].
[0301] Depression
[0302] There is a growing body of evidence that suggests the presence of ‘gut-brain’ axis and its significant role in anxiety and depression36–38. There are no known biomarkers [blood or genetic] for effectively diagnosing “Depression”. However, recent studies are leaning towards gut microbial markers that can indicate depression, perhaps at an earlier time, and aid in bi-directional rectification of gut microbiome for reducing depression39,40.
[0303] The BugSpeaks® cumulative risk scoring, estimated utilizing the evidence-based risk/safe tags for many microorganisms associated with Depression [from MicroByte] and its abundance [from Individual], has a continuous scale of 0-100. To estimate the sensitivity and specificity statistics, we utilized 20 sample data, of which 10 were diagnosed with Depression [as consented and disclosed by individuals] and 10 were Non-Depression controls. All data were received from patients with their informed consent.
Sample ID Diagnosed Risk Call Risk Score TP/FN
Sample_1 Depression Moderate 42 True Positive
Sample_2 Depression Moderate 56 True Positive
Sample_3 Depression Moderate 52 True Positive
Sample_4 Depression Moderate 43 True Positive
Sample_5 Depression Moderate 50 True Positive
Sample_6 Depression Moderate 50 True Positive
Sample_7 Depression Low 29 False Negative
Sample_8 Depression Moderate 60 True Positive
Sample_9 Depression Moderate 68 True Positive
Sample_10 Depression Low 8 False Negative
Total Samples
20
True Positive False Negative
8 2
Sensitivity

Table 5 – De-identified samples of patients with depression and Bugspeaks disease susceptibility call.
Sample ID Diagnosed Risk Call Risk Score TN/FP Total Samples
Sample_1 Non-Depression Low 25 True Negative 20
Sample_2 Non-Depression Low 36 True Negative True Negative False Positive
Sample_3 Non-Depression Low 35 True Negative 9 1
Sample_4 Non-Depression Low 39 True Negative Specificity

Sample_5 Non-Depression Low 38 True Negative
Sample_6 Non-Depression Low 27 True Negative
Sample_7 Non-Depression Low 30 True Negative
Sample_8 Non-Depression Low 31 True Negative
Sample_9 Non-Depression Low 30 True Negative
Sample_10 Non-Depression Moderate 53 False Positive

Table 6 – De-identified samples of control patients without depression and Bugspeaks disease susceptibility call.
[0304] Overall, based on the cutoff score of 40 or greater, we estimated the sensitivity of risk estimation of Depression by BugSpeaks® to be 0.80 [8/10 tests] and the specificity of the disease to be 0.90 [1/10 controls].
[0305] Although the experimental data provided above pertains to 3 specific diseases, we believe our risk estimation algorithm will be as sensitive and specific in estimating the risk of other diseases in the same category as those diseases [Ex. IBD representing the ability to estimate the risk of other FGIDs such as IBS, Constipation etc.].
[0306] ADVANTAGES OF THE INVENTION:
[0307] Provide recommendations of dietary, prebiotic and probiotic-centred approach to improve overall health and wellbeing
[0308] Compositions and dietary recommendations for maintaining or enhancing homeostasis or function
[0309] Prebiotic and probiotic recommendations for rectifying dysbiosis and related disease states or symptoms
[0310] May also be used as disease predictor (preventive healthcare) especially for functional gastrointestinal disorders (FGIDs)
[0311] Improving detoxification potential, immune responses and overall immune function, which are major components of wellness
a. Comprehensive analysis of Bacterial, Archaeal, Fungal, Viral, Metazoal and Protozoal composition and abundance of the gut microbiome (not limited to just Bacterial)
b. Health and wellness, rather than clinical. This removes those clinical barricades and focus turns to DTC process/product.
c. Since focus is health and wellness, the characterization of one's gut microbiome enable us to provide actionable recommendations, which will aid the individual in improving his/her overall health and wellbeing.
d. The actionable well-tolerated recommendations, which is the key differentiator, include:
i. 3 Phase dietary recommendation - with specific list of foods and frequency tags (frequent, moderate, low and avoid)
ii. Probiotic recommendations - to rejuvenate the gut microbiome, to promote gut microbial composition, with lasting effects on overall health.
iii. Prebiotic recommendations - to aid the rejuvenation process of the gut microbiome.
[0312] The second key differentiator is that these recommendations are strictly evidence based with reported associations with diseases, traits and overall health and wellbeing.
[0313] The third key differentiator is the ability to predict susceptibility to certain diseases based on the gut microflora (Appendix 3). This unique proposition will aid in better lifestyle management including but not limited to customized food intake (recommendation) also provided by this unique offering and thus leading to healthy living.
[0314] BEST MODE TO PRACTICE
[0315] The best mode to practice the invention is through commercialization of this invention through the distributors, e-commerce sites, and marketing to reach private healthcare and wellness clinics and hospitals.
[0316] According to aspects of the present invention, the invention also provides kits that are useful for the practice of the methods of the invention.
[0317] USES, APPLICATIONS, AND BENEFITS OF THE INVENTION
[0318] Direct to Consumer
[0319] End to end solution from registration to report
[0320] Overall, the innovation enables us to
a. Characterization of the microbiome
b. Provide recommendations of dietary, prebiotic, and probiotic-centred approach to improve overall health and wellbeing.
c. Compositions and dietary recommendations for maintaining or enhancing homeostasis or function.
d. Prebiotic and probiotic recommendations for rectifying dysbiosis and related disease states or symptoms
e. May also be used as disease predictor (preventive healthcare) especially for functional gastrointestinal disorders (FGIDs)
[0321] Improving detoxification potential, immune responses, and overall immune function, which are major components of wellness.
[0322] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0323] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0324] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0325] It should be understood that the figures and/or screenshots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0326] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

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,CLAIMS:CLAIMS

I/ We Claim,
1) A method for predicting disease susceptibility by profiling microbiota, comprising the steps of:
a) registration of user;
b) dispatching the kit;
c) obtaining sample comprising the gut microbiome from a human patient to be diagnosed;
d) extracting DNA;
e) sequencing DNA;
f) analyzing the data;
g) concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient;
h) predicting disease susceptibility; and
i) recommending a personalized nutritional regimen based on individual’s gut microbiota.
2) The method as claimed in claim 1, wherein the disease susceptibility is provided based on individual’s gut microbiota.
3) The method as claimed in claim 1 or 2, wherein said disease conditions are Irritable Bowel Syndrome, Colorectal Neoplasm, Constipation, Diabetes Mellitus Type 2, Inflammatory Bowel Disease, Non-Alcoholic Fatty Liver Disease, Ulcerative Colitis, Aerobic Endurance, Crohn’s Disease, Physical Endurance, Clostridium Difficile Infection, Power, Depression, Prone to Fatigue, Anxiety, Gluten intolerance, Rheumatoid Arthritis, Asthma, Chronic Kidney Disease, Eczema, Atherosclerosis, Atopic dermatitis, Obesity, Acne, Hypertension, Effect of Smoking, Parkinson’s Disease, Autism spectrum disorder, Alzheimer’s disease, Preeclampsia or any combination thereof.
4) A method for profiling microbiota, comprising the steps of:
[0327] obtaining raw data;
[0328] checking quality of data;
[0329] mapping on Microbial Database (MetaRef);
[0330] doing Taxonomical Profiling;
[0331] estimating Abundance Estimation;
[0332] plotting Generation;
[0333] analysing diversity;
[0334] mapping on MicroByte database;
[0335] estimating or detecting Disease Risk;
[0336] mapping on NutrBite database; and
[0337] recommending a personalized nutritional requirement.
5) The method as claimed in claim 4, wherein the method for analysing the microbiome comprises computational analysis on MetaRef, MicroByte, NutriBite and MetaRich data bases.
6) The method as claimed in claim 4, wherein the method for estimating the disease risks is based on microbiome of the individual.
7) The method as claimed in claim 4, wherein the said detecting comprises a method, such as a quantitative method, selected from the group of PCR, rtPCR, qPCR, multiplex PCR, high-throughput sequencing, metatranscriptomic sequencing, identification of strain-specific markers, such as genes and/or proteins, and 16S rDNA analysis.
8) A method for monitoring the progress of microbiome-based disease condition comprising the steps of:
a) obtaining sample comprising the gut microbiome from a human patient to be diagnosed
b) extracting DNA;
c) sequencing DNA;
d) analyzing the data;
e) concluding on microbiota in said patient, wherein an increase of the abundance of a said species as detected in said sample compared to a sample from a control patient;
f) monitoring the disease progress; and
g) recommending a personalized nutritional requirement.
9) A method for assessing the status of a disease, condition, pathology, or cell state, wherein the disease, condition, pathology, or cell state is associated with the presence or level of a cellular component, the method comprising the steps of detecting and/or isolating or removing the cellular component associated with the disease, condition, pathology, or cell state by a method according to any of Claims 1 or claim 4, and correlating the presence or level of the cellular component with disease, condition, pathology, or cell state.
10) A kit comprising:
a) Kit contents and guide;
b) A pair of hand gloves;
c) Stool collection frame;
d) Stool collection instructions;
e) Stool collection tube;
f) Protective bubble envelope with desiccant;
g) Questionnaire and informed consent;
h) Return Envelope; and
i) an instruction booklet.