Methods And Systems For Microbiome Characterization, Monitoring And Treatment

Abstract

The present disclosure provides methods for profiling a microbiome and therapeutic compositions for treatment. Additionally, the methods, systems, compositions and kits provided herein are directed to assessing or predicting health status in a subject. Some of the embodiments include generating a report.

Description

CROSS-REFERENCE

[0001] This application is a continuation of U.S. application Ser. No. 14/437,133, filed Apr. 20, 2015, now U.S. Pat. No. 10,633,714, which is a National Stage entry of PCT Application No. PCT/US2014/047491, filed on Jul. 21, 2014, which claims the benefit of U.S. Provisional Application No. 61/856,711 filed on Jul. 21, 2013, each of which is incorporated herein by reference in its entirety.

SEQUENCES

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 21, 2014, is named 46790-701.601_SL.txt and is 171,171 bytes in size

BACKGROUND OF THE DISCLOSURE

[0003] A typical healthy individual's body is inhabited with trillions of microbes across various body sites called microbiomes. Some examples of microbiome sites include skin, intestinal, stomach, gut, oral, conjunctival, and vaginal. To better understand the role of these microbiomes and how they affect physiology and disease state we can analyze what microbes comprise a microbiome and how they correlate or affect the health status and clinical response of an individual.

[0004] For example, the human gut microbiome is known to play a key role in many health conditions, including obesity, gastrointestinal health, nutrient absorption, and drug metabolism among others. Owing to such discoveries, the NIH has invested $150 million in the analysis of the Human Microbiome Project over the next 5-years for analyzing the microbial composition of various human body sites.

[0005] Despite this awareness of the interrelation between microbiomes and health, the complexity of the microbiome, as well as difficulties in categorizing and characterizing the constituents of the microbiome have made understanding these relationships challenging. Consequently, these challenges have presented hurdles in the development of diagnostic and therapeutic applications.

[0006] Metagenomic approaches to understanding the microbiome stand to help further illuminate the roles of the microbiomes and have only recently been enabled by "next-generation" sequencing technologies. While the information uncovered by these studies will become increasingly valuable to those interested in targeting the microbiome for therapeutic interventions and consumer products, transforming this large amount of data into meaningful data that can be used to develop diagnostics and therapeutics presents a significant hurdle. Two apparent bottlenecks in harnessing the power of the microbiome, is the cost of undertaking these analyses and the intrinsic complexity of metagenomic analysis mentioned above.

[0007] The current gold standard in the field for taxonomic classification of bacterial species is through the DNA sequencing of the 16S ribosomal RNA (rRNA) subunit. The 16S rRNA subunit was chosen as an "ideal" target for classification because it is universally present in all bacteria and it contains nine variable regions which can be used to distinguish taxonomies. However, focusing solely on the 16S rRNA subunit presents its own technical challenges owing to the fact that some bacteria share the same variable regions resulting in misclassification.

[0008] Furthermore, current "second-generation" sequencing technologies being used to sequence the 16S rRNA subunit have read lengths which often yield incomplete coverage of theses variable regions. For example, sequencing by 454 gives average read lengths of 500 bp and Illumina's MiSeq and HiSeq platforms give average read lengths of 100-450 bp. With these read lengths, bacterial classification often suffer from issues of accuracy, especially in a complex metagenomic sample such as a microbiome sample.

[0009] The present disclosure provides solutions to these limitations by providing methods, systems, compositions, and kits that yield more accurate information and hence more accurate classification of a microbiome. Such information allows for multiplex and efficiency advantages over the current technology and the development of consumer products such diagnostic tests, therapeutics and probiotic therapies.

SUMMARY OF THE INVENTION

[0010] The present disclosure provides a method of classifying a microbe, comprising: obtaining a nucleic acid sequence of a 16S or 23 S ribosomal subunit contained within a single read length of a first microbe; and comparing said nucleic acid sequence of a first microbe to a reference; and identifying the first microbe at the strain level or sub-strain level based on the comparing.

[0011] The present disclosure provides a method of profiling a microbiome in a subject, comprising: obtaining nucleic acids sequences of a 16S ribosomal subunit from at least one microbe in a biological sample obtained from the subject; analyzing said at least one microbe within said biological sample based upon the nucleic acids sequences obtained; and determining a profile of the microbiome based on said analyzing. In some embodiments, determining a profile of the microbiome in said subject can be based on 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbe, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewer microbes. In some embodiments determining a profile of the microbiome in said subject has an accuracy greater than 70% based on the measurements. In some embodiments, the method can further comprise obtaining nucleic acids sequences of from at least one microbe in a biological sample taken at least two different points of time. In some embodiments, analyzing uses long read sequencing platforms.

[0012] The present disclosure provides a method of profiling a microbiome in a subject, comprising: obtaining nucleic acids sequences of a 16S and 23S ribosomal subunit from at least one microbe in a biological sample obtained from said subject; analyzing said at least one microbe within said biological sample based upon the sequences obtained; and determining a profile of the microbiome in said subject based on said analyzing. In some embodiments, determining a profile of the microbiome in said subject can be based on 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbe, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewer microbes. In some embodiments, determining a profile of the microbiome in said subject has an accuracy greater than 70% based on the measurements. In some embodiments, the method can further comprising obtaining nucleic acids sequences of from at least one microbe in a biological sample taken at least two different points of time. In some embodiments, analyzing uses long read sequencing platforms.

[0013] The present disclosure provides a method of determining metabolic pathways that are indicative of a health status in a subject, comprising: obtaining RNA sequences from a biological sample from a subject, such that the entire transcript is contained within a single read length; analyzing said transcripts by a sequencing method; comparing the sequenced transcripts to a reference; and determining the metabolic pathways that are indicative of a health status. In some embodiments, analyzing uses long read sequencing platforms.

[0014] The present disclosure provides a method of treating a disease in a subject, comprising: measuring a microbiome profile in a biological sample obtained from the subject, wherein the microbiome profile comprises at least one microbe; detecting a presence or absence of the disease in the subject based upon said measuring; and treating the disease in the subject based upon said detecting.

[0015] The present disclosure provides a method, comprising: obtaining data comprising a measurement of a microbiome panel in a biological sample obtained from a subject, wherein said microbiome panel comprises at least two microbes; generating a microbiome profile of said microbiome panel based upon the measurement data; comparing said microbiome profile of said microbiome panel to a reference profile; and determining a likelihood of a disease status in said subject based said comparing.

[0016] The present disclosure provides a method, comprising: obtaining data comprising a measurement of a microbiome panel in a biological sample obtained from a subject, wherein said microbiome panel comprises at least two microbes; generating a microbiome profile of said microbiome panel based upon the measurement data; comparing said microbiome profile of said microbiome panel to a threshold level of a reference; and determining a likelihood of a disease status in said subject based on said comparing of at least one threshold level of a reference of said microbiome panel.

[0017] The present disclosure provides a method of diagnosing a subject a disease in a subject, comprising: measuring a microbe panel in a biological sample obtained from the subject, wherein the microbiome panel comprises at least one microbe; detecting a presence or absence of a disease state in said subject based upon said measuring; and; recommending to the subject at least one microbial-based therapeutic or cosmetic for treatment of said disease based on the detecting. In some embodiments, detecting a presence or absence of said disease state with a sensitivity that is greater than 70%. In some embodiments, detecting a presence or absence said disease state with a sensitivity and specificity that is greater than 70%. In some embodiments, comprising detecting a presence or absence said disease state with a sensitivity that is greater than 75%, 80%, 85%, 90%, or 95%. In some embodiments, detecting a presence or absence said disease state with a sensitivity and specificity that is greater than 75%, 80%, 85%, 90%, or 95%. In some embodiments, the panel of microbes comprise 2 or fewer microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, or 40 or fewer microbes.

[0018] The present disclosure provides a computer system for determining a microbiome profile in a subject, the computer system comprising: a memory unit for receiving data comprising measurement of a microbe panel from a biological sample of the subject, wherein the microbe panel comprises at least one marker of a microbe; computer-executable instructions for analyzing the measurement data according to a method of any of the preceding claims; and computer-executable instructions for determining a presence or absence of at least disease in the subject based upon said analyzing. In some embodiments, the computer system further comprises computer-executable instructions to generate a report of the presence or absence of the at least one disease in the subject. In some embodiments, computer system can further comprises a user interface configured to communicate or display said report to a user.

[0019] The present disclosure provides a computer readable medium comprising: computer-executable instructions for analyzing data comprising measurement of a microbiome profile from a biological sample obtained from a subject, wherein the microbiome profile comprises at least one marker selected from at least one microbe; and computer-executable instructions for determining a presence or absence of at least disease in the subject based upon the analyzing.

[0020] The present disclosure provides a kit, comprising: one or more compositions for use in measuring a microbiome profile in a biological sample obtained from a subject, wherein the microbiome profile comprises at least one marker to at least one microbe; and instructions for performing a method of any of the preceding claims. In some embodiments, a kit can further comprises a computer readable medium.

[0021] Any of the methods provided herein can include embodiments in wherein the disease is inflammatory bowel disease (IBD), preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type II diabetes, C. difficile, irritable bowel syndrome (IBS), asthma, autism, psoriasis, allergies, cardiovascular disease, cancer depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, type I diabetes, halitosis, and tooth decay.

[0022] Any of the methods provided herein can include embodiments wherein the biological sample is taken from a microbiome is selected from the group skin microbiome, umbilical microbiome, vaginal microbiome, conjunctival microbiome, intestinal microbiome, stomach microbiome, gut microbiome, oral microbiome, nasal microbiome, gastrointestinal tract microbiome, urogenital tract microbiome, or a combination thereof.

[0023] Any of the methods provided herein can include embodiments wherein the microbiome panel comprises a microbial marker of the 16S or 23S ribosomal subunit, or wherein the microbiome panel comprises a microbial marker to the entire 16S or 23S ribosomal subunit transcript, or wherein the microbiome panel comprises a microbial marker of the entire 16S or 23S ribosomal subunit transcript and the intergenic region between said 16S or 23S ribosomal subunit transcript, or wherein the microbiome panel comprises a microbial marker of at least one variable region of the 16S or 23S ribosomal subunit.

[0024] Any of the methods provided herein can include embodiments wherein the measuring comprises detecting or measuring a level of a fragment, antigen, or binding partner of the 16S or 23S ribosomal subunit, or wherein the measuring comprises detecting or measuring a level of a fragment, antigen, or binding partner of a 16S or 23S ribosomal subunit variable region.

[0025] Any of the methods provided herein can include embodiments wherein the subject is a human subject, or wherein the subject is asymptomatic for a disease, or wherein the subject is presenting with at least one clinical symptom for said disease.

[0026] Any of the methods provided herein can include embodiments wherein measuring comprises use of at least one of: an immunoassay, flow cytometry assay, biochip assay, microarray assay, and sequencing assay. Any of the methods provided herein can include embodiments wherein measuring comprises detecting or measuring a level of a microbe. Any of the methods provided herein can include embodiments wherein measuring comprises detecting or measuring a level of a marker on a microbe.

[0027] Any of the methods provided herein can include embodiments wherein the disease state determined is a poor clinical outcome, a good clinical outcome, a high risk of disease, a low risk of disease, a complete response, a partial response, a stable disease, a non-response, or a recommended treatments for disease management.

INCORPORATION BY REFERENCE

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0030] FIG. 1 depicts an exemplary computer system for implementing a method described herein. This includes a continually enlarging database of full rRNA operons as the methods described herein allow this to be expanded in a cost-effective manner that hasn't been previously available.

[0031] FIGS. 2A and 2B. FIG. 2A depicts the hypervariable regions of the 16s operon. FIG. 2B depicts simulation results in which the microbe classification accuracy (Y-axis) is plotted as a function of different 16S ribosomal amplicon target sizes. Bacterial sequences from the Ribosomal Database Project were used, with different sequencing platform error rates (X-axis). Utilizing the full 16S amplicon (top line) enables the highest classification accuracy for the practically realized error rates depicted (<5%).

[0032] FIG. 3 depicts changes in an individual's gut microbiome profile to changes in diet. In this example, the change in weight (dashed line) is a lagging indicator, when compared to the change in the microbiome structure. Subject-specific diet recommendation can be based on quantitative microbiome signatures.

[0033] FIGS. 4A and 4B depict that different genera have different locations of variation that would allow one to distinguish strains. This means that utilizing methods in which only a portion of 16S is observed would intrinsically limit the strain level classification accuracy. The boxplots on the bottom have an X-axis that corresponds to the method utilized (CBT corresponds to the full length 16S amplicon), and a Y-axis that corresponds to the percentage strain classification accuracy achieved. FIG. 4A depicts strain level resolution data with a method described herein for the Nitrosomonas genus, in which the per strain divergence is concentrated in the V3, 4, and 5 regions of 16S FIG. 4B depicts strain level resolution data with a method described herein for the Staphylococcus genus, in which the per strain divergence is concentrated in the V8, and 9 regions of 16S.

[0034] FIGS. 5A and 5B depict a study in which subjects (that include cases and controls for a specific indications as provided herein) apply a microbial therapeutic/cosmetic containing a consortia of strains. FIG. 5A depicts how the methods of this disclosure can be used to quantify the successful application of the microbial therapeutic/cosmetic over time. The addition of a non-commensal strain to the consortia, aids in distinguishing applied strains from the background variation of individual microbiomes for that site (e.g. skin, gut, mouth, etc.). FIG. 5B depicts how the methods in the present disclosure can be used to discover stabilizing commensal strains. Those strains that when present, are correlated to the longevity of the applied consortia, would become candidates for expanding the initial consortia to produce formulations with increased stability and efficacy.

[0035] FIGS. 6A and 6B. FIG. 6A depicts the resolution of a microbiome using current standard methods that resolve down to the genus level of a microbe comprising a microbiome FIG. 6B depicts the resolution of a microbiome using a method described herein that resolves down to the strain level of a microbe comprising a microbiome.

[0036] FIGS. 7A and 7B. FIG. 7A depicts a prophetic example in which usage of the standard approaches currently being used for genus level resolution. The results indicate that the standard approaches do not separate disease cases from healthy controls because the variation at the strain level is masked FIG. 7B depicts a prophetic example in which the disease cases are well separated from healthy controls using the methods provided herein.

[0037] FIG. 8. depicts a top ten list of microbial strains responsible for the distinguishing of healthy control and disease subjects depicted in Example 5, FIG. 7B. This information can be used for diagnostics and therapeutics for this indication.

DETAILED DESCRIPTION OF THE DISCLOSURE

I. Definitions

[0038] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0039] The term "microbes", "microorganisms" as used herein, refers to any single-celled organisms, bacteria, archaea, protozoa, and unicellular fungi.

[0040] The term "microbiome", as used herein, refers to the ecological community of commensal, symbiotic, or pathogenic microorganisms that inhabit a body space on a subject.

[0041] The terms "determining", "measuring", "evaluating", "assessing," "assaying," and "analyzing" can be used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. (e.g., detection). These terms can include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. These terms can include use of the algorithms and databases described herein. "Detecting the presence of" can include determining the amount of something present, as well as determining whether it is present or absent. The term "genome assembly algorithm" as used herein, refers to any method capable of aligning short reads with reference sequences under conditions that a complete sequence of the genome may be determined.

[0042] The term "genome" as used herein, refers to the entirety of an organism's hereditary information that is encoded in its primary DNA sequence. The genome includes both the genes and the non-coding sequences. For example, the genome may represent a microbial genome or a mammalian genome.

[0043] "Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

[0044] The terms "homology" and "homologous" as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., "substantially homologous," to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence.

[0045] The term "sequencing" as used herein refers to sequencing methods for determining the order of the nucleotide bases--adenine, guanine, cytosine, and thymine--in a nucleic acid molecule (e.g., a DNA or RNA nucleic acid molecule.

[0046] The term "biochip" or "array" can refer to a solid substrate having a generally planar surface to which an adsorbent is attached. A surface of the biochip can comprise a plurality of addressable locations, each of which location may have the adsorbent bound there. Biochips can be adapted to engage a probe interface, and therefore, function as probes. Protein biochips are adapted for the capture of polypeptides and can be comprise surfaces having chromatographic or biospecific adsorbents attached thereto at addressable locations. Microarray chips are generally used for DNA and RNA gene expression detection.

[0047] The term "barcode" as used herein, refers to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating genome of a nucleic acid fragment.

[0048] The terms "subject," "individual" or "patient" are used interchangeably herein. A "subject" can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microbe, including, e.g., bacteria, bacterial plasmids, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0049] The terms "treatment" or "treating" are used interchangeably herein. These terms can refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit can mean eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

Overview

[0050] The present disclosure is generally directed to the identification, classification or quantification of at least one microbiome comprising comprehensive analysis of at least one of the 16S and 23S ribosomal RNA (rRNA) subunits or intergenic regions.

[0051] The present disclosure also provides for the determination of a microbiome profile of a subject. The methods, compositions, systems and kit can generate a cohort-generalized microbiome profile or a subject-specific microbiome profile. The microbial profile can have an accuracy of 70% or greater based on measurement of 15 or fewer microbes in the biological sample. Such profiling method can have at least an accuracy greater than 70% based on measurement of no more than 2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, or 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbes, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewer microbes.

[0052] Such microbiome profile can be used in part or solely to calculate a quantitative score. A quantitative score can also use the microbiome profile in combination with one or more clinical factors such as age group, ethnicity, sexual habits, hygiene habits, product use, dietary regimen, weight, gender, medical history, risk factors, or family history.

[0053] In some embodiments, a quantitative score can indicate an increased or decreased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0054] In some applications, a decrease or increase in one or more microbes' threshold values in a subject's microbiome profile indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a decrease or increase in one or more microbes' threshold values in a subject's microbiome indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0055] Provided herein are microbial-based compositions that can be administered as therapeutic or as a cosmetic to a subject. Also provided herein are various formulations of the microbial-based compositions. One or more, or combinations of microbes or formulations provided herein can be used to develop appropriate compostions for treating a subject suffering from a condition. Any of the methods, compositions, kits, and systems described herein can be used to generate a therapeutic/cosmetics consumer product composition.

[0056] Any of the methods, compositions, kits, and systems described herein can be used to determine or predict disease status of a subject. Disease status can include such information as a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0057] The methods, compositions, systems and kits described herein provide a diagnostic assay for a disease, as described herein, based on detection or measurement of one or more microbes in a biological sample or a microbiome profile obtained from a subject. In some applications, the biological sample is collected from a healthy subject who wants to determine one or more of their microbiome profiles. In some applications, the biological sample collected from a subject with a disease who wants to determine one or more of their microbiome profiles. Indications that can be used with the methods, compositions, systems and kits as described herein include, but are not limited to IBD, preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type 2 diabetes, C. difficile, IBS, asthma, autism, psoriasis, allergies, cardiovascular disease, cancer, depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, type 1 diabetes, halitosis, and tooth decay.

[0058] A biological sample can be any sample type from any microbiome on the body of a subject. Some examples of microbiomes that can be used with the present disclosure include the skin microbiome, umbilical microbiome, vaginal microbiome, conjunctival microbiome, intestinal microbiome, stomach microbiome, gut microbiome and oral microbiome, nasal microbiome, gastrointestinal tract microbiome, and the urogenital tract microbiome. Depending on the application the biological sample can be whole blood, serum, plasma, mucosa, saliva, cheek swab, urine, stool, cells, tissue, bodily fluid or a combination thereof.

[0059] The diagnostic assays or methods provided herein can have at least one of a sensitivity of greater than 70% and specificity of greater than 70% of a disease status, prediction of disease response or outcome. Such diagnostic methods can have at least one of a sensitivity of 70% or greater and specificity of greater than 70% based on measurement of 15 or fewer microbes in the biological sample. Such diagnostic assays or method can have at least one of a sensitivity greater than 70% and specificity greater than 70% based on measurement of no more than 2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, or 100 or fewer microbes.

[0060] The methods, compositions, systems and kits described herein can be used to generate a report. In some applications, a report can include information such as the degree of likelihood (increase or decrease) of one or more of health status of a disease state: presence or absence of a disease state, a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0061] An exemplary method can comprise at least one of the following steps: obtaining a biological sample from a subject; measuring a panel of microbes in the biological sample of the subject; determining a disease status upon the measuring; and generating a report that provides information of disease status upon the results of the determining.

[0062] An exemplary method of the present disclosure can comprise at least one of the following steps: obtaining a biological sample from a subject, measuring a panel of microbes in the biological sample of the subject, determining a presence or absence at least one microbe in a subject's microbiome upon the measuring; generating a report that provides information on the absence or quantity of at least one microbe in a subject's microbiome profile upon determining, and optionally providing information from a cohort that has been determined to have a similar microbiome profile as the subject-specific microbiome profile.

[0063] The methods, compositions, systems and kits described herein also provide quality control assay for a manufactured microbial-based therapeutic/cosmetic. For example, the methods, compositions, systems and kits described herein can be used to develop companion diagnostic test to determine if a manufactured microbial-based therapeutic/cosmetic has maintained its genetic integrity during the manufacturing or storage process.

II. Methods

[0064] A. Microbiome Profiling

[0065] The present disclosure provides for methods for measuring at least one microbe in biological sample from at least one microbiome from a subject and determining a microbiome profile. A microbiome profile can be assessed using any suitable detection means that can measure or quantify one or more microbes (bacteria, fungi, viruses and archaea) that comprise a microbiome.

[0066] In general, the present disclosure employs long read length sequencing processes and systems to measure the full 16S or the 23S ribosomal subunits, their intergenic regions and optionally other genetic elements with discriminative power in a particular microbe in order to identify informative microbiome profiles.

[0067] In some applications, the microbial profile of a subject is in part determine using additional clinical information such as the subject's age, weight, gender, medical history, risk factors, family history or any other clinically relevant information.

[0068] In some applications, a subject's microbiome profile can comprise a single microbiome. For example, a subject's microbiome profile can comprise of at least one biological sample from only the subject's intestinal microbiome. For example, a subject's microbiome profile can comprise of at least one biological sample from only the subject's stomach microbiome. For example, a subject's microbiome profile can comprise of at least one biological sample from only the subject's gut microbiome. For example, a subject's microbiome profile can comprise of at least one biological sample from only the subject's oral microbiome.

[0069] In some applications, a subject's microbiome profile can comprise of at least one biological sample from more than one microbiome. For example, a subject's microbiome profile can comprise of at least one biological sample from the subject's skin microbiome and umbilical microbiome. In another example, a subject's microbiome profile can comprise of at least one biological sample from the subject's intestinal microbiome, stomach microbiome, gut microbiome and oral microbiome. In another example, a subject's microbiome profile can comprise of at least one biological sample from the subject's intestinal microbiome and at least one biological sample from stomach microbiome. In another example, a subject's microbiome profile can comprise of at least one biological sample from the subject's gut microbiome and at least one biological sample from oral microbiome. In some applications, a subject's microbiome profile can comprise of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 microbiomes.

[0070] In some applications, a subject's microbiome profile can comprise of one microbe. In some applications, a subject's microbiome profile can comprise of 2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbe, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewer microbes.

[0071] In some applications the entire genome to the microbe will be analyzed to determine a subject's microbiome profile. In other applications, the variable regions of the microbe's genome will be analyzed to determine a subject's microbiome profile. For example genetic variation in the genome can include restriction fragment length polymorphisms, single nucleotide polymorphisms, insertions, deletions, indels (insertions-deletions), microsatellite repeats, minisatellite repeats, short tandem repeats, transposable elements, randomly amplified polymorphic DNA, amplification fragment length polymorphism or a combination thereof.

[0072] In some applications, the entire genomic region of the 16S or 23S ribosomal subunit to the microbe will be analyzed to determine a subject's microbiome profile. In some applications, the variable regions of the 16S or 23S ribosomal subunit to the microbe will be analyzed to determine a subject's microbiome profile.

[0073] The methods, compositions, systems provided herein can generate a cohort-generalized microbiome profile or a subject-specific microbiome profile. A cohort-generalized microbiome profile can be determined upon the measurement data from more than one subject's microbiome from a particular group. For example, a cohort, can be subjects from a particular age group, region of the world, ethnicity, religious group, sexual habits, hygiene habits, product use, dietary regimen, weight, gender, medical history, risk factors, family history, or combinations thereof. A subject-specific microbiome profile can be determined from one or more microbiomes from a subject.

[0074] An exemplary method can comprise at least one of the following steps: obtaining a biological sample from a subject; measuring at least one microbe in the biological sample of the subject; detecting or measuring the presence or absence of at least one microbe upon measuring; and generating a report that provides details the presence, absence, or quantity of at least one microbe in a subject's microbiome.

[0075] An exemplary method can comprise at least one of the following steps: obtaining a biological sample from a subject; measuring a panel of microbes in the biological sample of the subject; detecting a presence or absence of the panel of microbes upon measuring; determining the subject's microbiome profile; and generating a report that provides details about the determined microbiome profile or a similar microbiome profile.

[0076] The present disclosure provides diagnostic assays for predicting a disease status of a subject or likelihood of a subject's response to a therapeutic. The diagnostic assay can use the presence of one or more microbes to calculate a quantitative score that can be used to predict disease status or likelihood of response to a therapeutic in a subject. In some applications, the diagnostic assay can use the presence of one or more microbes and one or more characteristics, such as, e.g., age, weight, gender, medical history, risk factors, family history, or a combination thereof to calculate a quantitative score that can be used to predict disease status or likelihood of response to a therapeutic in a subject.

[0077] In some applications, a decrease in one or more microbes' threshold values in a subject's microbiome profile indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some embodiments, a decrease in the quantitative score indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0078] In some applications, a decrease in one or more microbes' threshold values in a subject's microbiome profile indicates a decreased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some embodiments, a decrease in the quantitative score indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0079] In some applications, an increase in one or more microbes' threshold values in a subject's microbiome profile indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a decrease in one or more microbes' threshold values indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0080] In some applications, an increase in one or more microbes' threshold values in a subject's microbiome profile indicates a decreased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a decrease in one or more microbes' threshold values indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0081] In some applications, a similar microbiome profile to a reference profile indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a dissimilar microbiome profile to a reference profile indicates one or more of: an increased likelihood of a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0082] In some applications, a similar microbiome profile to a reference profile indicates a decreased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a dissimilar microbiome profile to a reference profile indicates one or more of: an increased likelihood of a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0083] B. Samples

[0084] Biological samples can be collected from a subject who wants information on one or more of their microbiomes. Any sample type from any microbiome on the body of a subject can be used with the methods, systems, and kits of the present disclosure. Examples of microbiomes that can be used with the present disclosure include but are not limited to skin microbiome, umbilical microbiome, vaginal microbiome, conjunctival microbiome, intestinal microbiome, stomach microbiome, gut microbiome and oral microbiome, nasal microbiome, gastrointestinal tract microbiome, and urogenital tract microbiome.

[0085] Depending on the application the selection of a biological sample may be tailored to that specific application. In any of the methods provided herein the biological sample can be whole blood, serum, plasma, mucosa, saliva, cheek swab, urine, stool, cells, tissue, lymph fluid, CNS fluid, and lesion exudates or a combination thereof.

[0086] C. Sample Preparation

[0087] Biological samples used with the methods, composition, systems, and kits provide hererin may be processed using any means known in the art or otherwise described herein in order to enable measurement of one or more microbes. Sample preparation can comprise any one of the following steps or combination of steps: 1) a sterile swab is first dipped into a tube containing sterile 1.times.PBS to wet. 2) The swab is swiped across the area of interest 10-20 times with enough vigor that the tissue is slightly pink/red colored afterwards. 3) The swab is gently dipped into 300 uL of lysis buffer (described herein) in a sterile 1.5 mL tube. 4) The swab is left in the microcentrifuge tube for shipping to a laboratory to be further analyzed as provided herein. The samples obtained can be shipped overnight at room temperature.

[0088] Shipping bacterial cells in buffers have inherent biases associated with them--some strains are able to continue propagating on the very few nutrients that come along with sample collection while other strains will undergo apoptosis in the absence of a very specific environment. As a result, samples shipped in this fashion often have an initial profiling/population bias associated with cellular integrity.

[0089] Current approaches strongly enrich for intact cells by first centrifuging the collected sample. The resulting pellet, formed from the intact cells within the sample, is then the precursor for all of the downstream steps. In contrast, the present methods provided herein includes a purification step to concentrate any DNA present in the supernatant (e.g. from already lysed cells). This DNA is then combined with the DNA extracted from the standard pellet preparation, and this combination now forms the more complete precursor to the downstream steps.

[0090] In the present methods provided herein, microbiome samples can be immediately put into the specific lysis buffer cocktail described below, rather than standard buffers are stable for 3 days at room temperature, allowing enough time for samples that need to be shipped. These samples can then be processed as usual and DNA will remain intact for use in long readlength sequencing and microbiome profiling. The Lysis buffer contains: 20 mM Tris, pH8.0, 20 mM EDTA, 1% SDS, 0.5% Tween, 1% Triton X-100, and 400 ug/mL proteinase K.

[0091] The subsequent extraction of DNA from human microbiome samples includes several steps, each of which contribute to sample integrity and artifactual chimeric molecule production. For the amplicon approach these steps can include: PCR, sample quantification (e.g. Qubit, nanodrop, bioanalyzer, etc.), Blue Pippin size selection, 0.5.times. Ampure purification, sample quantification, DNA end repair, 0.5.times. Ampure purification, blunt end adaptor ligation, exo-nuclease treatment, two 0.5.times. Ampure purifications, and final Blue Pippen size selection. Depending on the sample one or more of these steps maybe removed to improve the fidelity of the microbiome profiling. This is determined empirically in a feed-back loop with the profiling, by using known mock communities as tests, or by introducing known quantities of non-commensal strains into the sample.

[0092] In some applications, the method does not use an amplification step. Examples of such methods include those methods that use sequencing by Whole Genome Shotgun (WGS) sequencing. These approaches can provide a benefit by removing amplification bias, which is known to skew microbial distributions. In addition, such approaches also allows for de novo discovery of pertinent elements, for example such as bacterial plasmids, fungi and viruses.

[0093] The practice of the methods of the present disclosure can employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE AND SPECIALIZED APPLICATIONS, 6th Edition (R. I. Freshney, ed. (2010), and Lange, et. al., Molecular Systems Biology Vol. 4:Article 222 (2008), which are hereby incorporated by reference. For example, preparation of a biological sample may comprise, e.g., extraction or isolation of intracellular material from a cell or tissue such as the extraction of nucleic acids, protein, or other macromolecules. Sample preparation which can be used with the methods of disclosure include but are not limited to, centrifugation, affinity chromatography, magnetic separation, immunoassay, nucleic acid assay, receptor-based assay, cytometric assay, colorimetric assay, enzymatic assay, electrophoretic assay, electrochemical assay, spectroscopic assay, chromatographic assay, microscopic assay, topographic assay, calorimetric assay, radioisotope assay, protein synthesis assay, histological assay, culture assay, and combinations thereof.

[0094] Accessing the nucleic acids and macromolecules from the intercellular space of the sample may generally be performed by either physical, chemical methods, or a combination of both.

[0095] The nucleic acids used with the methods described herein can be isolated from any biological samples using shearing methods which preserve the integrity and continuity of genomic DNA.

[0096] Nucleic acids sample that can be used with the present disclosure include all types of DNA and RNA. The length of nucleic acids can be about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, or 10,000,000, nucleotides or base pairs in length.

[0097] Variations found in the nucleic acids "variable regions" may provide a means for distinguishing genomes or strains. Examples of such variation in the nucleic acids include but are not limited to, polymorphisms such as: restriction fragment length polymorphisms, single nucleotide polymorphisms, insertions, deletions, indels (insertions-deletions), microsatellite repeats, minisatellite repeats, short tandem repeats, transposable elements, randomly amplified polymorphic DNA, and amplification fragment length polymorphism.

[0098] D. Detection

[0099] Profiling of a microbiome can be conducted by various means known in the art, some of which are provided herein. In some applications, microbiome profiling can comprise one or more detection means.

[0100] In some applications samples will be measured using sequencing methods. In certain application the PCR primers, provided herein, will contain unique barcode identifiers. In some applications the addition of the barcode identifiers allows for multiplexing of multiple samples (e.g. biological samples or microbiome samples) using the computers systems and executable-code provide herein. In applications where single molecule sequencing is used, computers systems and executable-code that can derive base modification detection for each sample, as provided herein, can be useful.

[0101] Examples sequencing technologies that can be used with the present methods, systems and kits of the disclosure for achieving long read lengths include but are not limited to the SMRT.RTM. sequencing systems from Pacific Biosciences. In particular, the SMRT systems produce average read lengths in excess of 5000 bases in length, based upon single molecule, real time observation of nucleic acid replication (See, e.g., U.S. Pat. Nos. 7,056,661, 7,056,676, 7,052,847 and 7,033,764, and Chin C S, et. al., Nonhybrid finished microbial genome assemblies from long read SMRT sequencing data. Nat Methods. 2013 June; 10(6): 563-9)

[0102] Other sequencing systems and approaches that can be used with the present disclosure include but are not limited to long read length Sanger sequencing, long read ensemble sequencing approaches, e.g., Illumina/Moleculo sequencing and potentially, other single molecule sequencing approaches, such as Nanopore sequencing technologies.

[0103] In applications where long read sequencing is used with the methods of the present disclosure, long read sequencing can include sequencing that provides a contiguous sequence read of longer than 500 bases, preferably, longer than 800 bases, more preferably, greater than 1000 bases, and in most preferred aspects, longer than 1500 bases. Long read sequencing can also include sequencing that provides a contiguous sequence read of longer than 1500 bases, preferably, longer than 2000 bases, more preferably, greater than 3000 bases, and longer than 4500 bases.

[0104] In particularly preferred aspects, single molecule, real time sequencing approaches are preferred, such as the Pacific Biosciences SMRT technology. In particular, by providing both extremely long read lengths and single molecule resolution, these systems provide advantages for classification accuracy as well as the potential for extremely high multiplex, e.g., with different individual molecules providing different opportunities to classify different constituents within a microbiome sample.

[0105] Because each single molecule read spans the entire 16S and/or 23S region, assembly is not required. Therefore, barcoding and multiplexing can be employed to reduce the cost per strain/sample.

[0106] Suitable barcodes that can be used with the invention include but are not limited to SEQ ID NOS 1-16 and SEQ ID NOS 660-742.

[0107] The present disclosure takes advantage of the long read length, single molecule sequencing system in order to classify microbial organisms in a mixed population. The resulting "microbiome profile" will take advantage of the outputs, which contain the entire 16S and 23S regions, to achieve unparalleled classification accuracy at a lower cost.

[0108] Additionally the present invention includes a non-amplification mode in which a whole genome shotgun (WGS) sample is used to profile the microbiome. This removes amplification bias from the profiling which is known to be problematic. Reads from any part of the meta-genome can thus be used as the strain identifier. This allows for de novo discovery of pertinent elements such as bacterial plasmids, fungi and viruses. This also expands the possible level of variation to be observed since the requirement of known constant regions is removed. By comparing the results of the amplicon approach with WGS, biases in the amplicon approach can be corrected and a sample efficient, amplicon version, can be created for specific applications. This also allows the usage of base modification signal since the sample preparation does not include an amplification step.

[0109] In addition to read length and multiplex benefits, single molecule real time sequencing is also useful for obtaining information on base modifications present in a given organism's genome. In particular, by using long read length technologies which are sensitive to base modifications, the disclosure can take advantage of this additional or 5.sup.th base data to add classification specificity to sample analyses and classification. Because DNA modifications are known to affect gene expression, it is clear that microbes, such as bacteria with the same genome but different base modifications should be considered different strains with distinct activities/host interactions.

[0110] In addition to applying the read length and multiplex advantages towards improving phylogenetic classification using 16S, 23S, or other genomic locations, these advantages can also be used towards a fuller delineation of the true expression profile of the microbiome. Thus, full length transcript sequencing will yield a more complete picture of what metabolic pathways are most relevant for improving host health.

[0111] Microbiome profiling can comprise usage of a nucleic acid microarray. The microbiome can be measured in either fresh or fixed sample using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs, RNA, mRNA, ect. and oligonucleotides) can be plated, or arrayed, on a microchip substrate. The arrayed sequences can be then hybridized with specific probes with complementarity to the oligonucleotides on the substrate. The arrayed sequences can also be PCR amplified inserts of nucleic acid clones can be applied to a substrate in a dense array.

[0112] In some applications, there can be greater than 100, 500, 1,000, 2000, 3,000 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 nucleotide sequences can be applied to the substrate. The microarrayed genes, variable regions, intergenic regions or other regions of interest are immobilized on the microchip at greater than 100, 500, 1,000, 2000, 3,000 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 elements each, can be suitable for hybridization under stringent conditions.

[0113] Fluorescently labeled probes may be generated through incorporation of fluorescent nucleotides. Labeled probes can then be applied to the chip hybridize with specificity to each spot of the array. After stringent washing to remove non-specifically bound probes, the microarray chip can be scanned by a device such as, confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding abundance. With dual color fluorescence, separately labeled probes generated from more than one source of nucleic acids can be hybridized to the array. The relative abundance of the nucleic acids sources corresponding to each specified gene can be thus determined. Analysis of the microarray readout can be performed by commercially available equipment.

[0114] Microbiome profiling can further comprise of use of a biochip. Biochips can be used to screen a large number of macromolecules. Biochips can be designed with immobilized nucleic acid molecules, full-length proteins, antibodies, affibodies (small molecules engineered to mimic monoclonal antibodies), aptamers (nucleic acid-based ligands) or chemical compounds. A chip could be designed to detect multiple macromolecule types on one chip. For example, a chip could be designed to detect nucleic acid molecules, proteins and metabolites on one chip. The biochip can be used to and designed to simultaneously analyze a panel microbes in a single sample.

[0115] In some applications, microbiome profiling can comprise use of a protein microarray. Protein microarray can be a particular type of biochip which can be used with the present disclosure. The chip can comprise a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins can be bound in an arrayed format onto a solid surface. Protein array detection methods can give a high signal and a low background. Detection probe molecules, typically labeled with a fluorescent dye, can be added to the array. Any reaction between the probe and the immobilized protein can result in emission of a detectable signal. Such protein microarrays can be rapid, automated, and offer high sensitivity of protein markers known to be located on a microbe read-outs for diagnostic tests.

[0116] In some applications, microbiome profiling can comprise use of an analytical protein microarrays can be constructed using a library of antibodies, aptamers or affibodies. The array can be probed with a complex protein solution from a biological sample that function by capturing protein molecules they specifically bind to. Analysis of the resulting binding reactions using various detection systems can provide information about expression levels of particular proteins in the sample as well as measurements of binding affinities and specificities. This type of protein microarray can be especially useful in comparing protein expression in different samples. Functional protein microarrays can be constructed by immobilizing large numbers of purified full-length functional proteins or protein domains and can be used to identify protein-protein, protein-DNA, protein-RNA, protein-phospholipid, and protein-small molecule interactions, to assay enzymatic activity and to detect antibodies and demonstrate their specificity. These protein microarray biochips can be used to study the biochemical activities of the entire proteome in a sample.

[0117] In some applications, microbiome profiling can comprise use of reverse phase protein microarray (RPA). Reverse phase protein microarray can be constructed from tissue and cell lysates that can be arrayed onto the microarray and probed with antibodies against the target protein of interest. These antibodies can be detected with chemiluminescent, fluorescent or colorimetric assays. In addition to the protein in the lysate, reference control peptides can be printed on the slides to allow for protein quantification.

[0118] In some applications, microbiome profiling can further comprise use of a digital PCR device or droplet digital PCR device. Droplet digital PCR can be used to partition molecules such as DNA, RNA or protein in a biological sample to a compartment and identifying and measure molecules in that compartment. Interrogation of each droplet can yield counts and measurements of molecules present in the biological sample.

[0119] E. Primers and Probes

[0120] The analysis of the 16S ribosomal RNA gene is one approach that can be used to understand microbial diversity. Another approach that can be applied is the analysis of the 23S ribosomal RNA gene. The accuracy of these analyses depends strongly on the choice of primers.

[0121] Primers can be prepared by a variety of methods including, but not limited to, cloning of appropriate sequences and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)). Primers can also be obtained from commercial sources such as Integrated DNA Technologies, Operon Technologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies. In addition, computer programs can also be used to design primers, including but not limited to Array Designer Software (Arrayit Inc.), Oligonucleotide Probe Sequence Design Software for Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis from Hitachi Software Engineering.

[0122] Primers that can be used analyze the 16S ribosomal RNA gene include but are not limited to SEQ ID NOS 17-24.

[0123] Primers that can be used analyze the 16S ribosomal RNA gene include but are not limited to SEQ ID NOS 17-24 and 646-656.

[0124] Primers that can be used analyze the 23S ribosomal RNA gene include but are not limited to SEQ ID NOS 25-67 and 657-659.

[0125] Microbial diversity can be further described by approaches analyzing the intergenic region between 16S ribosomal RNA and 23S ribosomal RNA. Primers that can be used to analyze the intergenic region between 16S ribosomal RNA and 23S ribosomal RNA include but are not limited to SEQ ID NOS 270-364 (forward intergenic primers) and 551-645 (reverse intergenic primers).

[0126] Primers that can be designed to specifically amplify and identified variable regions in the 16S ribosomal RNA and 23S ribosomal RNA include but are not limited to SEQ ID NOS 87-180 (forward 16S primers), 181-269 (forward 23S primers), 365-461 (reverse 16S primers) and 462-550 (reverse 23S primers). Primers can be designed to specifically amplify any identified variable regions in a microbe or similar distinguishing genetic element.

[0127] Primers or probes described herein can also include polynucleotides having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any of the nucleic acid sequences described herein.

[0128] Primers or probes described herein can also include polypeptide having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to any of the nucleic acid sequences described herein.

[0129] In some applications, it may be useful to incorporate restriction sites into primer or probe sequence depending on the particular application. Examples of restriction enzymes that can be used with the methods of the present discloure include but are not limited to: AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrlI, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsalI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnlI, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI, FspI, HaelI, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinPlI, HpaI, HpaII, HphI, Hpyl66II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAlI, MspI, MwoI, NaeI, NacI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, Pad, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqaI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI, and ZraI.

[0130] In some applications, it may be useful to incorporate a barcode into primer or probe sequence depending on the particular application. Examples of barcodes that can be used with the invention are provided herein. Barcoding of the biological sample can be used to facilitate multiplexing assays as provided herein. Barcoding of the biological sample can be used in methods described herein that use sequencing as the detection means.

[0131] F. Algorithm-Based Methods

[0132] The present disclosure provides for machine learning algorithms for building a diagnostic microbiome profile of a subject. Depending on the application a diagnostic microbiome profile can a generate score from a microbiome profile, can be a comparison to a reference microbiome profile, can be the level of a microbiome profile above a defined threshold or a combination thereof.

[0133] The present disclosure includes sequencing methods that utilize higher error rate, single molecule platforms. These systems are asynchronous in nature, thus providing the long readlengths necessary to properly classify the complex strains that make up the microbiome.

[0134] In the case of amplicon sequencing (16S, 23S, and other marker genes) the raw data produced from this platform is first filtered for proper primer orientation, pairing, and completeness. The resulting molecules are then filtered based on quality (with quality thresholds of greater than 0.95, 0.99, 0.999, etc. being possible). These molecules then form the basis set of reads to be used to establish the de novo clusters, and can directly be compared to the known reference databases. Molecules that only partially match to the known reference database are appended to a novel hit database. Stringent read length thresholds of >1,000 bases prevent spurious reads from mistakenly making it into the novel hit database. An empirically (using mock community or non-commensal introduced strain) determined error weighting is used in the clustering to minimize the effect of platform specific sequencing artifacts affecting the clustering.

[0135] In the case of WGS, empirically determined (using mock community or non-commensal introduced strain) cutoff read lengths and accuracies are used and those are matched to a maximum sensitivity threshold. The resulting molecules are then filtered based on quality (with quality thresholds of greater than 0.95, 0.99, 0.999, etc. being possible). These molecules then form the basis set of reads to be used to establish the de novo clusters, and can directly be compared to the known reference databases. Molecules that only partially match to the known reference database are appended to a novel hit database. Stringent read length thresholds of >1,000 bases prevent spurious reads from mistakenly making it into the novel hit database. An empirically (using mock community or non-commensal introduced strain) determined error weighting is used in the clustering to minimize the effect of platform specific sequencing artifacts affecting the clustering.

[0136] Examples of machine learning algorithms that can be used include, but are not limited to: elastic networks, random forests, support vector machines, and logistic regression. The algorithms provided herein can aid in selection of important microbes and transform the underlying measurements into a score or probability relating to, for example, disease risk, disease likelihood, presence or absence of disease, treatment response, and/or classification of disease status.

[0137] Any of the methods, kits, and systems described herein can utilize a diagnostic assay for predicting a disease status of a subject or likelihood of a subject's response to a therapeutic. The diagnostic assay can use the presence of one or more microbes to calculate a quantitative score that can be used to predict disease status or likelihood of response to a therapeutic in a subject. The diagnostic assay can use the presence of one or more microbes and one or more characteristics, such as, e.g., age, weight, gender, medical history, risk factors, family history to calculate a quantitative score that can be used to predict disease status or likelihood of response to a therapeutic in a subject.

[0138] In some applications, an increase in a score in the diagnostic assay indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some embodiments, a decrease in the quantitative score indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0139] In some applications, a decrease in a score in the diagnostic assay indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some embodiments, a decrease in the quantitative score indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0140] In some applications, a similar microbiome profile to a reference profile in the diagnostic assay indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a dissimilar microbiome profile to a reference profile indicates one or more of: an increased likelihood of a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0141] In some applications, an increase in one or more microbes' threshold values in the diagnostic assay indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a decrease in one or more microbes threshold values indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0142] In some applications, a decrease in one or more microbes' threshold values in the diagnostic assay indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a decrease in one or more microbes threshold values indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0143] The present disclosure provides methods of treatments. Provided herein are methods for generalized-treatment recommendations for a subject based on their microbiome profiling and methods for subject-specific treatment recommendation. Methods for treatments can comprise one of the following steps: determining a first ratio of a level of a subject-specific microbiome profile to a level of a second microbiome profile in a biological sample obtained from at least one subject; detecting a presence or absence of a disease in the subject based upon the determining; and recommending to the subject at least one generalized or subject-specific treatment to ameliorate disease symptoms.

[0144] Any dignostic microbiome profile, a subject-specific microbiome profile, or a therapeutic/cosmetic described herein can include one or more, but are not limited to the following microbes: Abiotrophia, Abiotrophia defectiva, Abiotrophia, Acetanaerobacterium, Acetanaerobacterium elongatum, Acetanaerobacterium, Acetivibrio, Acetivibrio bacterium, Acetivibrio, Acetobacterium, Acetobacterium, Acetobacterium woodii, Acholeplasma, Acholeplasma, Acidaminococcus, Acidaminococcus fermentans, Acidaminococcus, Acidianus, Acidianus brierleyi, Acidianus, Acidovorax, Acidovorax, Acinetobacter, Acinetobacter guillouiae, Acinetobacter junii, Acinetobacter, Actinobacillus, Actinobacillus M1933/96/1, Actinomyces, Actinomyces ICM34, Actinomyces ICM41, Actinomyces ICM54, Actinomyces lingnae, Actinomyces odontolyticus, Actinomyces oral, Actinomyces ph3, Actinomyces, Adlercreutzia, Adlercreutzia equolifaciens, Adlercreutzia intestinal, Adlercreutzia, Aerococcus, Aerococcus, Aeromonas, Aeromonas 165C, Aeromonas hydrophila, Aeromonas RC50, Aeromonas, Aeropyrum, Aeropyrum pernix, Aeropyrum, Aggregatibacter, Aggregatibacter, Agreia, Agreia bicolorata, Agreia, Agromonas, Agromonas C S30, Akkermansia, Akkermansia muciniphila, Akkermansia, Alistipes, Alistipes ANH, Alistipes AP11, Alistipes bacterium, Alistipes CCUG, Alistipes DJF_B185, Alistipes DSM, Alistipes EBA6-25c12, Alistipes finegoldii, Alistipes indistinctus, Alistipes JC136, Alistipes NMLO5A004, Alistipes onderdonkii, Alistipes putredinis, Alistipes RMA, Alistipes senegalensis, Alistipes shahii, Alistipes Smarlab, Alistipes, Alkalibaculum, Alkalibaculum, Alkaliflexus, Alkaliflexus, Allisonella, Allisonella histaminiformans, Allisonella, Alloscardovia, Alloscardovia omnicolens, Anaerofilum, Anaerofilum, Anaerofustis, Anaerofustis stercorihominis, Anaerofustis, Anaeroplasma, Anaeroplasma, Anaerostipes, Anaerostipes 08964, Anaerostipes 1y-2, Anaerostipes 494a, Anaerostipes 5_1_63FAA, Anaerostipes AIP, Anaerostipes bacterium, Anaerostipes butyraticus, Anaerostipes caccae, Anaerostipes hadrum, Anaerostipes Anaerostipes indolis, Anaerostipes, Anaerotruncus, Anaerotruncus colihominis, Anaerotruncus NML, Anaerotruncus, Aquincola, Aquincola, Arcobacter, Arcobacter, Arthrobacter, Arthrobacter FV1-1, Asaccharobacter, Asaccharobacter celatus, Asaccharobacter, Asteroleplasma, Asteroleplasma, Atopobacter, Atopobacter phocae, Atopobium, Atopobium parvulum, Atopobium rimae, Atopobium, Bacteriovorax, Bacteriovorax, Bacteroides, Bacteroides 31SF18, Bacteroides 326-8, Bacteroides 35AE31, Bacteroides 35AE37, Bacteroides 35BE34, Bacteroides 4072, Bacteroides 7853, Bacteroides acidifaciens, Bacteroides AP1, Bacteroides AR20, Bacteroides AR29, Bacteroides B2, Bacteroides bacterium, Bacteroides barnesiae, Bacteroides BLBE-6, Bacteroides BV-1, Bacteroides caccae, Bacteroides CannelCatfish9, Bacteroides cellulosilyticus, Bacteroides chinchillae, Bacteroides CIP103040, Bacteroides clams, Bacteroides coprocola, Bacteroides coprophilus, Bacteroides D8, Bacteroides DJF_B097, Bacteroides dnLKV2, Bacteroides dnLKV7, Bacteroides dnLKV9, Bacteroides dorei, Bacteroides EBA5-17, Bacteroides eggerthii, Bacteroides enrichment, Bacteroides F-4, Bacteroides faecichinchillae, Bacteroides faecis, Bacteroides fecal, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides gallinamm, Bacteroides helcogenes, Bacteroides ic1292, Bacteroides intestinalis, Bacteroides massiliensis, Bacteroides mpnisolate, Bacteroides NB-8, Bacteroides new, Bacteroides nlaezlc13, Bacteroides nlaezlc158, Bacteroides nlaezlc159, Bacteroides nlaezlc161, Bacteroides nlaezlc163, Bacteroides nlaezlc167, Bacteroides nlaezlc172, Bacteroides nlaezlc18, Bacteroides nlaezlc182, Bacteroides nlaezlc190, Bacteroides nlaezlc198, Bacteroides nlaezlc204, Bacteroides nlaezlc205, Bacteroides nlaezlc206, Bacteroides nlaezlc207, Bacteroides nlaezlc211, Bacteroides nlaezlc218, Bacteroides nlaezlc257, Bacteroides nlaezlc260, Bacteroides nlaezlc261, Bacteroides nlaezlc263, Bacteroides nlaezlc308, Bacteroides nlaezlc315, Bacteroides nlaezlc322, Bacteroides nlaezlc324, Bacteroides nlaezlc331, Bacteroides nlaezlc339, Bacteroides nlaezlc36, Bacteroides nlaezlc367, Bacteroides nlaezlc375, Bacteroides nlaezlc376, Bacteroides nlaezlc380, Bacteroides nlaezlc391, Bacteroides nlaezlc459, Bacteroides nlaezlc484, Bacteroides nlaezlc501, Bacteroides nlaezlc504, Bacteroides nlaezlc515, Bacteroides nlaezlc519, Bacteroides nlaezlc532, Bacteroides nlaezlc557, Bacteroides nlaezlc57, Bacteroides nlaezlc574, Bacteroides nlaezlc592, Bacteroides nlaezlg105, Bacteroides nlaezlg117, Bacteroides nlaezlg127, Bacteroides nlaezlg136, Bacteroides nlaezlg143, Bacteroides nlaezlg157, Bacteroides nlaezlg167, Bacteroides nlaezlg171, Bacteroides nlaezlg187, Bacteroides nlaezlg194, Bacteroides nlaezlg195, Bacteroides nlaezlg199, Bacteroides nlaezlg209, Bacteroides nlaezlg212, Bacteroides nlaezlg213, Bacteroides nlaezlg218, Bacteroides nlaezlg221, Bacteroides nlaezlg228, Bacteroides nlaezlg234, Bacteroides nlaezlg237, Bacteroides nlaezlg24, Bacteroides nlaezlg245, Bacteroides nlaezlg257, Bacteroides nlaezlg27, Bacteroides nlaezlg285, Bacteroides nlaezlg288, Bacteroides nlaezlg295, Bacteroides nlaezlg296, Bacteroides nlaezlg303, Bacteroides nlaezlg310, Bacteroides nlaezlg312, Bacteroides nlaezlg327, Bacteroides nlaezlg329, Bacteroides nlaezlg336, Bacteroides nlaezlg338, Bacteroides nlaezlg347, Bacteroides nlaezlg356, Bacteroides nlaezlg373, Bacteroides nlaezlg376, Bacteroides nlaezlg380, Bacteroides nlaezlg382, Bacteroides nlaezlg385, Bacteroides nlaezlg4, Bacteroides nlaezlg422, Bacteroides nlaezlg437, Bacteroides nlaezlg454, Bacteroides nlaezlg455, Bacteroides nlaezlg456, Bacteroides nlaezlg458, Bacteroides nlaezlg459, Bacteroides nlaezlg46, Bacteroides nlaezlg461, Bacteroides nlaezlg475, Bacteroides nlaezlg481, Bacteroides nlaezlg484, Bacteroides nlaezlg5, Bacteroides nlaezlg502, Bacteroides nlaezlg515, Bacteroides nlaezlg518, Bacteroides nlaezlg521, Bacteroides nlaezlg54, Bacteroides nlaezlg6, Bacteroides nlaezlg8, Bacteroides nlaezlg80, Bacteroides nlaezlg98, Bacteroides nlaezlh120, Bacteroides nlaezlh15, Bacteroides nlaezlh162, Bacteroides nlaezlh17, Bacteroides nlaezlh174, Bacteroides nlaezlh18, Bacteroides nlaezlh188, Bacteroides nlaezlh192, Bacteroides nlaezlh194, Bacteroides nlaezlh195, Bacteroides nlaezlh207, Bacteroides nlaezlh22, Bacteroides nlaezlh250, Bacteroides nlaezlh251, Bacteroides nlaezlh28, Bacteroides nlaezlh313, Bacteroides nlaezlh319, Bacteroides nlaezlh321, Bacteroides nlaezlh328, Bacteroides nlaezlh334, Bacteroides nlaezlh390, Bacteroides nlaezlh391, Bacteroides nlaezlh414, Bacteroides nlaezlh416, Bacteroides nlaezlh419, Bacteroides nlaezlh429, Bacteroides nlaezlh439, Bacteroides nlaezlh444, Bacteroides nlaezlh45, Bacteroides nlaezlh46, Bacteroides nlaezlh462, Bacteroides nlaezlh463, Bacteroides nlaezlh465, Bacteroides nlaezlh468, Bacteroides nlaezlh471, Bacteroides nlaezlh472, Bacteroides nlaezlh474, Bacteroides nlaezlh479, Bacteroides nlaezlh482, Bacteroides nlaezlh49, Bacteroides nlaezlh493, Bacteroides nlaezlh496, Bacteroides nlaezlh497, Bacteroides nlaezlh499, Bacteroides nlaezlh50, Bacteroides nlaezlh531, Bacteroides nlaezlh535, Bacteroides nlaezlh8, Bacteroides nlaezlp104, Bacteroides nlaezlp105, Bacteroides nlaezlp108, Bacteroides nlaezlp132, Bacteroides nlaezlp133, Bacteroides nlaezlp151, Bacteroides nlaezlp157, Bacteroides nlaezlp166, Bacteroides nlaezlp167, Bacteroides nlaezlp171, Bacteroides nlaezlp178, Bacteroides nlaezlp187, Bacteroides nlaezlp191, Bacteroides nlaezlp196, Bacteroides nlaezlp208, Bacteroides nlaezlp213, Bacteroides nlaezlp228, Bacteroides nlaezlp233, Bacteroides nlaezlp267, Bacteroides nlaezlp278, Bacteroides nlaezlp282, Bacteroides nlaezlp286, Bacteroides nlaezlp295, Bacteroides nlaezlp299, Bacteroides nlaezlp301, Bacteroides nlaezlp302, Bacteroides nlaezlp304, Bacteroides nlaezlp317, Bacteroides nlaezlp319, Bacteroides nlaezlp32, Bacteroides nlaezlp332, Bacteroides nlaezlp349, Bacteroides nlaezlp35, Bacteroides nlaezlp356, Bacteroides nlaezlp370, Bacteroides nlaezlp371, Bacteroides nlaezlp376, Bacteroides nlaezlp395, Bacteroides nlaezlp402, Bacteroides nlaezlp403, Bacteroides nlaezlp409, Bacteroides nlaezlp412, Bacteroides nlaezlp436, Bacteroides nlaezlp438, Bacteroides nlaezlp440, Bacteroides nlaezlp447, Bacteroides nlaezlp448, Bacteroides nlaezlp451, Bacteroides nlaezlp476, Bacteroides nlaezlp478, Bacteroides nlaezlp483, Bacteroides nlaezlp489, Bacteroides nlaezlp493, Bacteroides nlaezlp557, Bacteroides nlaezlp559, Bacteroides nlaezlp564, Bacteroides nlaezlp565, Bacteroides nlaezlp572, Bacteroides nlaezlp573, Bacteroides nlaezlp576, Bacteroides nlaezlp591, Bacteroides nlaezlp592, Bacteroides nlaezlp631, Bacteroides nlaezlp633, Bacteroides nlaezlp696, Bacteroides nlaezlp7, Bacteroides nlaezlp720, Bacteroides nlaezlp730, Bacteroides nlaezlp736, Bacteroides nlaezlp737, Bacteroides nlaezlp754, Bacteroides nlaezlp759, Bacteroides nlaezlp774, Bacteroides nlaezlp828, Bacteroides nlaezlp854, Bacteroides nlaezlp860, Bacteroides nlaezlp886, Bacteroides nlaezlp887, Bacteroides nlaezlp900, Bacteroides nlaezlp909, Bacteroides nlaezlp913, Bacteroides nlaezlp916, Bacteroides nlaezlp920, Bacteroides nlaezlp96, Bacteroides nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides paurosaccharolyticus, Bacteroides plebeius, Bacteroides R6, Bacteroides rodentium, Bacteroides S-17, Bacteroides S-18, Bacteroides salyersiae, Bacteroides SLC1-38, Bacteroides Smarlab, Bacteroides 'Smarlab, Bacteroides stercorirosoris, Bacteroides stercoris, Bacteroides str, Bacteroides thetaiotaomicron, Bacteroides TP-5, Bacteroides, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides WA1, Bacteroides WH2, Bacteroides WH302, Bacteroides WH305, Bacteroides XB12B, Bacteroides XB44A, Bacteroides XO77B42, Bacteroides xylanisolvens, Barnesiella, Barnesiella intestinihominis, Barnesiella NSB1, Barnesiella, Barnesiella viscericola, Bavariicoccus, Bavariicoccus, Bdellovibrio, Bdellovibrio oral, Bergeriella, Bergeriella, Bifidobacterium, Bifidobacterium 103, Bifidobacterium 108, Bifidobacterium 113, Bifidobacterium 120, Bifidobacterium 138, Bifidobacterium 33, Bifidobacterium Acbbto5, Bifidobacterium adolescentis, Bifidobacterium Amsbbt12, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bacterium, Bifidobacterium bifidum, Bifidobacterium Bisn6, Bifidobacterium Bma6, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium choerinum, Bifidobacterium coryneforme, Bifidobacterium dentium, Bifidobacterium DJF WC44, Bifidobacterium F-10, Bifidobacterium F-11, Bifidobacterium group, Bifidobacterium h12, Bifidobacterium HMLN1, Bifidobacterium HMLN12, Bifidobacterium HMLN5, Bifidobacterium iarfr2341d, Bifidobacterium iarfr642d48, Bifidobacterium ic1332, Bifidobacterium indicum, Bifidobacterium kashiwanohense, Bifidobacterium LISLUCIII-2, Bifidobacterium longum, Bifidobacterium M45, Bifidobacterium merycicum, Bifidobacterium minimum, Bifidobacterium MSXSB, Bifidobacterium oral, Bifidobacterium PG12A, Bifidobacterium PL1, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium pullorum, Bifidobacterium ruminantium, Bifidobacterium S-10, Bifidobacterium saeculare, Bifidobacterium saguini, Bifidobacterium scardovii, Bifidobacterium simiae, Bifidobacterium SLPYG-1, Bifidobacterium stellenboschense, Bifidobacterium stercoris, Bifidobacterium TM-7, Bifidobacterium Trm9, Bifidobacterium, Bilophila, Bilophila nlaezlh528, Bilophila, Bilophila wadsworthia, Blautia, Blautia bacterium, Blautia CE2, Blautia CE6, Blautia coccoides, Blautia DJF VR52, Blautia DJF VR67, Blautia DJF VR70k1, Blautia formate, Blautia glucerasea, Blautia hansenii, Blautia ic1272, Blautia IES, Blautia K-1, Blautia luti, Blautia M-1, Blautia mpnisolate, Blautia nlaezlc25, Blautia nlaezlc259, Blautia nlaezlc51, Blautia nlaezlc520, Blautia nlaezlc542, Blautia nlaezlc544, Blautia nlaezlh27, Blautia nlaezlh316, Blautia nlaezlh317, Blautia obeum, Blautia producta, Blautia productus, Blautia schinkii, Blautia Ser5, Blautia Ser8, Blautia, Blautia WAL, Blautia wexlerae, Blautia YHC-4, Brenneria, Brenneria, Brevibacterium, Brevibacterium, Brochothrix, Brochothrix thermosphacta, Buttiauxella, Buttiauxella 57916, Buttiauxella gaviniae, Butyricicoccus, Butyricicoccus bacterium, Butyricicoccus, Butyricimonas, Butyricimonas 180-3, Butyricimonas 214-4, Butyricimonas bacterium, Butyricimonas GD2, Butyricimonas synergistica, Butyricimonas, Butyricimonas virosa, Butyrivibrio, Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Butyrivibrio, Caldimicrobium, Caldimicrobium, Caldisericum, Caldisericum, Campylobacter, Campylobacter coli, Campylobacter hominis, Campylobacter, Capnocytophaga, Capnocytophaga, Carnobacterium, Carnobacterium alterfunditum, Carnobacterium, Caryophanon, Caryophanon, Catenibacterium, Catenibacterium mitsuokai, Catenibacterium, Catonella, Catonella, Caulobacter, Caulobacter, Cellulophaga, Cellulophaga, Cellulosilyticum, Cellulosilyticum, Cetobacterium, Cetobacterium, Chelatococcus, Chelatococcus, Chlorobium, Chlorobium, Chryseobacterium, Chryseobacterium A1005, Chryseobacterium KJ9C8, Chryseobacterium, Citrobacter, Citrobacter 1, Citrobacter agglomerans, Citrobacter amalonaticus, Citrobacter ascorbata, Citrobacter bacterium, Citrobacter BinzhouCLT, Citrobacter braakii, Citrobacter enrichment, Citrobacter F24, Citrobacter F96, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter HBKC SR1, Citrobacter HD4.9, Citrobacter hormaechei, Citrobacter 191-3, Citrobacter ka55, Citrobacter lapagei, Citrobacter LAR-1, Citrobacter ludwigii, Citrobacter MEB5, Citrobacter MS36, Citrobacter murliniae, Citrobacter nlaezlc269, Citrobacter P014, Citrobacter P042bN, Citrobacter P046a, Citrobacter P073, Citrobacter SR3, Citrobacter T1, Citrobacter tnt4, Citrobacter tnt5, Citrobacter trout, Citrobacter TSA-1, Citrobacter, Citrobacter werkmanii, Cloacibacillus, Cloacibacillus adv66, Cloacibacillus nlaezlp702, Cloacibacillus NML05A017, Cloacibacillus, Cloacibacterium, Cloacibacterium, Collinsella, Collinsella A-1, Collinsella aerofaciens, Collinsella AUH-Julong21, Collinsella bacterium, Collinsella CCUG, Collinsella, Comamonas, Comamonas straminea, Comamonas testosteroni, Conexibacter, Conexibacter, Coprobacillus, Coprobacillus bacterium, Coprobacillus cateniformis, Coprobacillus TM-40, Coprobacillus, Coprococcus, Coprococcus 14505, Coprococcus bacterium, Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Coprococcus nexile, Coprococcus, Coraliomargarita, Coraliomargarita fucoidanolyticus, Coraliomargarita marisflavi, Coraliomargarita, Corynebacterium, Corynebacterium amycolatum, Corynebacterium durum, Coxiella, Coxiella, Cronobacter, Cronobacter dublinensis, Cronobacter sakazakii, Cronobacter turicensis, Cryptobacterium, Cryptobacterium curtum, Cupriavidus, Cupriavidus eutropha, Dechloromonas, Dechloromonas HZ, Desulfobacterium, Desulfobacterium, Desulfobulbus, Desulfobulbus, Desulfopila, Desulfopila

La4.1, Desulfovibrio, Desulfovibrio D4, Desulfovibrio desulfuricans, Desulfovibrio DSM12803, Desulfovibrio enrichment, Desulfovibrio fairfieldensis, Desulfovibrio LNB1, Desulfovibrio piger, Desulfovibrio, Dialister, Dialister E2_20, Dialister GBA27, Dialister invisus, Dialister oral, Dialister succinatiphilus, Dialister, Dorea, Dorea auhjulong64, Dorea bacterium, Dorea formicigenerans, Dorea longicatena, Dorea mpnisolate, Dorea, Dysgonomonas, Dysgonomonas gadei, Dysgonomonas, Edwardsiella, Edwardsiella tarda, Eggerthella, Eggerthella E1, Eggerthella lenta, Eggerthella MLGO43, Eggerthella MVA1, Eggerthella S6-C1, Eggerthella SDG-2, Eggerthella sinensis, Eggerthella str, Eggerthella, Enhydrobacter, Enhydrobacter, Enterobacter, Enterobacter 1050, Enterobacter 1122, Enterobacter 77000, Enterobacter 82353, Enterobacter 9C, Enterobacter ASC, Enterobacter adecarboxylata, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter AJAR-A2, Enterobacter amnigenus, Enterobacter asburiae, Enterobacter B1(2012), Enterobacter B363, Enterobacter B509, Enterobacter bacterium, Enterobacter Badong3, Enterobacter BEC441, Enterobacter C8, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter CO, Enterobacter core2, Enterobacter cowanii, Enterobacter dc6, Enterobacter DRSBII, Enterobacter enrichment, Enterobacter FL13-2-1, Enterobacter GIST-NKst10, Enterobacter GIST-NKst9, Enterobacter GJ1-11, Enterobacter gx-148, Enterobacter hormaechei, Enterobacter I-Bh20-21, Enterobacter ICB113, Enterobacter kobei, Enterobacter KW14, Enterobacter112, Enterobacter ludwigii, Enterobacter M10_1B, Enterobacter M1R3, Enterobacter marine, Enterobacter NCCP-167, Enterobacter of, Enterobacter oryzae, Enterobacter oxytoca, Enterobacter P101, Enterobacter S11, Enterobacter SEL2, Enterobacter SPh, Enterobacter SSASP5, Enterobacter terrigena, Enterobacter TNT3, Enterobacter TP2MC, Enterobacter T S4, Enterobacter TSSAS2-48, Enterobacter, Enterobacter ZYXCA1, Enterococcus, Enterococcus 020824/02-A, Enterococcus 1275b, Enterococcus 16C, Enterococcus 48, Enterococcus 6114, Enterococcus ABRIINW-H61, Enterococcus asini, Enterococcus avium, Enterococcus azikeevi, Enterococcus bacterium, Enterococcus BBDP57, Enterococcus BPH34, Enterococcus Bt, Enterococcus canis, Enterococcus casseliflavus, Enterococcus CmNA2, Enterococcus Da-20, Enterococcus devriesei, Enterococcus di spar, Enterococcus DJF 030, Enterococcus DMB4, Enterococcus durans, Enterococcus enrichment, Enterococcus F81, Enterococcus faecalis, Enterococcus faecium, Enterococcus fcc9, Enterococcus fecal, Enterococcus flavescens, Enterococcus fluvialis, Enterococcus FR-3, Enterococcus FUA3374, Enterococcus gallinarum, Enterococcus GHAPRB1, Enterococcus GSC-2, Enterococcus GYPB01, Enterococcus hermanniensis, Enterococcus hirae, Enterococcus lactis, Enterococcus malodoratus, Enterococcus manure, Enterococcus marine, Enterococcus MNC1, Enterococcus moraviensis, Enterococcus MS2, Enterococcus mundtii, Enterococcus NAB15, Enterococcus NBRC, Enterococcus nlaezlc434, Enterococcus nlaezlg106, Enterococcus nlaezlg87, Enterococcus nlaezlh339, Enterococcus nlaezlh375, Enterococcus nlaezlh381, Enterococcus nlaezlh383, Enterococcus nlaezlh405, Enterococcus nlaezlp116, Enterococcus nlaezlp148, Enterococcus nlaezlp401, Enterococcus nlaezlp650, Enterococcus pseudoavium, Enterococcus R-25205, Enterococcus raffinosus, Enterococcus rottae, Enterococcus RU07, Enterococcus saccharolyticus, Enterococcus saccharominimus, Enterococcus sanguinicola, Enterococcus SCA16, Enterococcus SCA2, Enterococcus SE138, Enterococcus SF-1, Enterococcus sulfureus, Enterococcus SV6, Enterococcus te1a, Enterococcus te32a, Enterococcus te42a, Enterococcus te45r, Enterococcus te49a, Enterococcus te51a, Enterococcus te58r, Enterococcus te59r, Enterococcus te61r, Enterococcus te93r, Enterococcus te95a, Enterococcus, Enterorhabdus, Enterorhabdus caecimuris, Enterorhabdus, Erwinia, Erwinia agglomerans, Erwinia enterica, Erwinia rhapontici, Erwinia tasmaniensis, Erwinia, Erysipelotrichaceae_incertae_sedis, Erysipelotrichaceae_incertae_sedis aff, Erysipelotrichaceae_incertae_sedis bacterium, Erysipelotrichaceae_incertae_sedis biforme, Erysipelotrichaceae_incertae_sedis C-1, Erysipelotrichaceae_incertae_sedis cylindroides, Erysipelotrichaceae_incertae_sedis GK12, Erysipelotrichaceae_incertae_sedis innocuum, Erysipelotrichaceae_incertae_sedis nlaezlc332, Erysipelotrichaceae_incertae_sedis nlaezlc340, Erysipelotrichaceae_incertae_sedis nlaezlg420, Erysipelotrichaceae_incertae_sedis nlaezlg425, Erysipelotrichaceae_incertae_sedis nlaezlg440, Erysipelotrichaceae_incertae_sedis nlaezlg463, Erysipelotrichaceae_incertae_sedis nlaezlh340, Erysipelotrichaceae_incertae_sedis nlaezlh354, Erysipelotrichaceae_incertae_sedis nlaezlh379, Erysipelotrichaceae_incertae_sedis nlaezlh380, Erysipelotrichaceae_incertae_sedis nlaezlh385, Erysipelotrichaceae_incertae_sedis nlaezlh410, Erysipelotrichaceae_incertae_sedis tortuosum, Erysipelotrichaceae_incertae_sedis, Escherichia/Shigella, Escherichia/Shigella 29(2010), Escherichia/Shigella 4091, Escherichia/Shigella 4104, Escherichia/Shigella 8gw18, Escherichia/Shigella A94, Escherichia/Shigella albertii, Escherichia/Shigella B-1012, Escherichia/Shigella B4, Escherichia/Shigella bacterium, Escherichia/Shigella BBDP15, Escherichia/Shigella BBDP80, Escherichia/Shigella boydii, Escherichia/Shigella carotovorum, Escherichia/Shigella CERAR, Escherichia/Shigella coli, Escherichia/Shigella DBC-1, Escherichia/Shigella dc262011, Escherichia/Shigella dysenteriae, Escherichia/Shigella enrichment, Escherichia/Shigella escherichia, Escherichia/Shigella fecal, Escherichia/Shigella fergusonii, Escherichia/Shigella flexneri, Escherichia/Shigella GDR05, Escherichia/Shigella GDR07, Escherichia/Shigella H7, Escherichia/Shigella marine, Escherichia/Shigella ML2-46, Escherichia/Shigella mpnisolate, Escherichia/Shigella NA, Escherichia/Shigella nlaezlg330, Escherichia/Shigella nlaezlg400, Escherichia/Shigella nlaezlg441, Escherichia/Shigella nlaezlg506, Escherichia/Shigella nlaezlh204, Escherichia/Shigella nlaezlh208, Escherichia/Shigella nlaezlh209, Escherichia/Shigella nlaezlh213, Escherichia/Shigella nlaezlh214, Escherichia/Shigella nlaezlh4, Escherichia/Shigella nlaezlh435, Escherichia/Shigella nlaezlh81, Escherichia/Shigella nlaezlp126, Escherichia/Shigella nlaezlp198, Escherichia/Shigella nlaezlp21, Escherichia/Shigella nlaezlp235, Escherichia/Shigella nlaezlp237, Escherichia/Shigella nlaezlp239, Escherichia/Shigella nlaezlp25, Escherichia/Shigella nlaezlp252, Escherichia/Shigella nlaezlp275, Escherichia/Shigella nlaezlp280, Escherichia/Shigella nlaezlp51, Escherichia/Shigella nlaezlp53, Escherichia/Shigella nlaezlp669, Escherichia/Shigella nlaezlp676, Escherichia/Shigella nlaezlp717, Escherichia/Shigella nlaezlp731, Escherichia/Shigella nlaezlp826, Escherichia/Shigella nlaezlp877, Escherichia/Shigella nlaezlp884, Escherichia/Shigella NMU-ST2, Escherichia/Shigella oc182011, Escherichia/Shigella of, Escherichia/Shigella proteobacterium, Escherichia/Shigella Q1, Escherichia/Shigella sakazakii, Escherichia/Shigella SF6, Escherichia/Shigella sm1719, Escherichia/Shigella SOD-7317, Escherichia/Shigella sonnei, Escherichia/Shigella SW86, Escherichia/Shigella, Escherichia/Shigella vulneris, Ethanoligenens, Ethanoligenens harbinense, Ethanoligenens, Eubacterium, Eubacterium ARC-2, Eubacterium callanderi, Eubacterium E-1, Eubacterium G3(2011), Eubacterium infirmum, Eubacterium limosum, Eubacterium methylotrophicum, Eubacterium nlaezlp439, Eubacterium nlaezlp457, Eubacterium nlaezlp458, Eubacterium nlaezlp469, Eubacterium nlaezlp474, Eubacterium oral, Eubacterium saphenum, Eubacterium sulci, Eubacterium, Eubacterium WAL, Euglenida, Euglenida longa, Faecalibacterium, Faecalibacterium bacterium, Faecalibacterium canine, Faecalibacterium DJF VR20, Faecalibacterium ic1379, Faecalibacterium prausnitzii, Faecalibacterium, Filibacter, Filibacter globispora, Flavobacterium, Flavobacterium SSL03, Flavobacterium, Flavonifractor, Flavonifractor AUH-JLC235, Flavonifractor enrichment, Flavonifractor nlaezlc354, Flavonifractor orbiscindens, Flavonifractor plautii, Flavonifractor, Francisella, Francisella piscicida, Fusobacterium, Fusobacterium nucleatum, Fusobacterium, Gardnerella, Gardnerella, Gardnerella vaginalis, Gemmiger, Gemmiger DJF VR33k2, Gemmiger formicilis, Gemmiger, Geobacter, Geobacter, Gordonibacter, Gordonibacter bacterium, Gordonibacter intestinal, Gordonibacter pamelaeae, Gordonibacter, Gp2, Gp2, Gp21, Gp21, Gp4, Gp4, Gp6, Gp6, Granulicatella, Granulicatella adiacens, Granulicatella enrichment, Granulicatella oral, Granulicatella paraadiacens, Granulicatella, Haemophilus, Haemophilus, Hafnia, Hafnia 3-12(2010), Hafnia alvei, Hafnia CC16, Hafnia proteus, Hafnia, Haliea, Haliea, Hallella, Hallella seregens, Hallella, Herbaspirillum, Herbaspirillum 022S4-11, Herbaspirillum seropedicae, Hespellia, Hespellia porcina, Hespellia stercorisuis, Hespellia, Holdemania, Holdemania AP2, Holdemania filiformis, Holdemania, Howardella, Howardella, Howardella ureilytica, Hydrogenoanaerobacterium, Hydrogenoanaerobacterium saccharovorans, Hydrogenophaga, Hydrogenophaga bacterium, Ilumatobacter, Ilumatobacter, Janthinobacterium, Janthinobacterium C30An7, Janthinobacterium, Jeotgalicoccus, Jeotgalicoccus, Klebsiella, Klebsiella aerogenes, Klebsiella bacterium, Klebsiella E1L1, Klebsiella EB2-THQ, Klebsiella enrichment, Klebsiella F83, Klebsiella G1-6, Klebsiella gg160e, Klebsiella granulomatis, Klebsiella HaNA20, Klebsiella HF2, Klebsiella ii_3_chl_1, Klebsiella KALAICIBA17, Klebsiella kpu, Klebsiella M3, Klebsiella MB45, Klebsiella milletis, Klebsiella NCCP-138, Klebsiella ok1_1_9_S16, Klebsiella ok1_1_9_S54, Klebsiella planticola, Klebsiella pneumoniae, Klebsiella poinarii, Klebsiella PSB26, Klebsiella RS, Klebsiella Se14, Klebsiella SRC DSD12, Klebsiella td153s, Klebsiella TG-1, Klebsiella TPS5, Klebsiella, Klebsiella variicola, Klebsiella WB-2, Klebsiella Y9, Klebsiella zlmy, Kluyvera, Kluyvera An5-1, Kluyvera cryocrescens, Kluyvera, Kocuria, Kocuria 2216.35.31, Kurthia, Kurthia, Lachnobacterium, Lachnobacterium C12b, Lachnobacterium, 0, Lachnospiracea_incertae_sedis bacterium, Lachnospiracea_incertae_sedis contortum, Lachnospiracea_incertae_sedis Eg2, Lachnospiracea_incertae_sedis eligens, Lachnospiracea_incertae_sedis ethanolgignens, Lachnospiracea_incertae_sedis galacturonicus, Lachnospiracea_incertae_sedis gnavus, Lachnospiracea_incertae_sedis hallii, Lachnospiracea_incertae_sedis hydrogenotrophica, Lachnospiracea_incertae_sedis ID5, Lachnospiracea_incertae_sedis intestinal, Lachnospiracea_incertae_sedis mpnisolate, Lachnospiracea_incertae_sedis pectinoschiza, Lachnospiracea_incertae_sedis ramulus, Lachnospiracea_incertae_sedis rectale, Lachnospiracea_incertae_sedis RLB1, Lachnospiracea_incertae_sedis rumen, Lachnospiracea_incertae_sedis SY8519, Lachnospiracea_incertae_sedis torques, Lachnospiracea_incertae_sedis, Lachnospiracea_incertae_sedis uniforme, Lachnospiracea_incertae_sedis ventriosum, Lachnospiracea_incertae_sedis xylanophilum, Lachnospiracea_incertae_sedis ye62, Lactobacillus, Lactobacillus 5-1-2, Lactobacillus 66c, Lactobacillus acidophilus, Lactobacillus arizonensis, Lactobacillus B5406, Lactobacillus brevis, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hominis, Lactobacillus ID9203, Lactobacillus IDSAc, Lactobacillus intestinal, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus manihotivorans, Lactobacillus mucosae, Lactobacillus NA, Lactobacillus oris, Lactobacillus P23, Lactobacillus P8, Lactobacillus paracasei, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus rennanqilfy10, Lactobacillus rennanqilfy14, Lactobacillus rennanqily19, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactobacillus suntoryeus, Lactobacillus T3R1C1, Lactobacillus, Lactobacillus vaginalis, Lactobacillus zeae, Lactococcus, Lactococcus 56, Lactococcus CR-317S, Lactococcus CW-1, Lactococcus D8, Lactococcus Da-18, Lactococcus DAP39, Lactococcus delbrueckii, Lactococcus F116, Lactococcus fujiensis, Lactococcus G22, Lactococcus garvieae, Lactococcus lactis, Lactococcus manure, Lactococcus RTS, Lactococcus SXVIII1(2011), Lactococcus TP2MJ, Lactococcus TP2ML, Lactococcus TP2MN, Lactococcus U5-1, Lactococcus, Lactonifactor, Lactonifactor bacterium, Lactonifactor longoviformis, Lactonifactor nlaezlc533, Lactonifactor, Leclercia, Leclercia, Lentisphaera, Lentisphaera, Leuconostoc, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc garlicum, Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc inhae, Leuconostoc lactis, Leuconostoc MEBE2, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc, Limnobacter, Limnobacter spf3, Luteolibacter, Luteolibacter bacterium, Lutispora, Lutispora, Marinifilum, Marinifilum, Marinobacter, Marinobacter arcticus, Mariprofundus, Mariprofundus, Marvinbryantia, Marvinbryantia, Megamonas, Megamonas, Megasphaera, Megasphaera, Melissococcus, Melissococcus faecalis, Methanobacterium, Methanobacterium subterraneum, Methanobrevibacter, Methanobrevibacter arboriphilus, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanobrevibacter oralis, Methanobrevibacter SM9, Methanobrevibacter smithii, Methanobrevibacter, Methanosphaera, Methanosphaera stadtmanae, Methanosphaera, Methylobacterium, Methylobacterium adhaesivum, Methylobacterium bacterium, Methylobacterium iEII3, Methylobacterium MP3, Methylobacterium oryzae, Methylobacterium PB132, Methylobacterium PB20, Methylobacterium PB280, Methylobacterium PDD-23b-14, Methylobacterium radiotolerans, Methylobacterium SKJH-1, Methylobacterium, Mitsuokella, Mitsuokella jalaludinii, Mitsuokella, Morganella, Morganella morganii, Morganella, Moritella, Moritella 2D2, Moryella, Moryella indoligenes, Moryella naviforme, Moryella, Mycobacterium, Mycobacterium tuberculosis, Mycobacterium, Negativicoccus, Negativicoccus, Nitrosomonas, Nitrosomonas eutropha, Novosphingobium, Novosphingobium, Odoribacter, Odoribacter laneus, Odoribacter splanchnicus, Odoribacter, Olsenella, Olsenella 1832, Olsenella F0206, Olsenella, Orbus, Orbus gilliamella, Oribacterium, Oribacterium, Oscillibacter, Oscillibacter bacterium, Oscillibacter enrichment, Oscillibacter, Owenweeksia, Owenweeksia, Oxalobacter, Oxalobacter formigenes, Oxalobacter, Paludibacter, Paludibacter, Pantoea, Pantoea agglomerans, Pantoea eucalypti, Pantoea, Papillibacter, Papillibacter cinnamivorans, Papillibacter, Parabacteroides, Parabacteroides ASF519, Parabacteroides CR-34, Parabacteroides distasonis, Parabacteroides DJF_B084, Parabacteroides DJF_B086, Parabacteroides dnLKV8, Parabacteroides enrichment, Parabacteroides fecal, Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae, Parabacteroides mpnisolate, Parabacteroi

des nlaezlp340, Parabacteroides, Paraeggerthella, Paraeggerthella hongkongensis, Paraeggerthella nlaezlp797, Paraeggerthella nlaezlp896, Paraprevotella, Paraprevotella clara, Paraprevotella, Paraprevotella xylaniphila, Parasutterella, Parasutterella excrementihominis, Parasutterella, Pectobacterium, Pectobacterium carotovorum, Pectobacterium wasabiae, Pediococcus, Pediococcus te2r, Pediococcus, Pedobacter, Pedobacter b3N1b-b5, Pedobacter daechungensis, Pedobacter, Peptostreptococcus, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Peptostreptococcus, Phascolarctobacterium, Phascolarctobacterium faecium, Phascolarctobacterium, Photobacterium, Photobacterium MIE, Pilibacter, Pilibacter, Planctomyces, Planctomyces, Planococcaceae_incertae_sedis, Planococcaceae_incertae_sedis, Planomicrobium, Planomicrobium, Plesiomonas, Plesiomonas, Porphyrobacter, Porphyrobacter KK348, Porphyromonas, Porphyromonas asaccharolytica, Porphyromonas bennonis, Porphyromonas canine, Porphyromonas somerae, Porphyromonas, Prevotella, Prevotella bacterium, Prevotella BI-42, Prevotella bivia, Prevotella buccalis, Prevotella copri, Prevotella DJF_B112, Prevotella mpnisolate, Prevotella oral, Prevotella, Propionibacterium, Propionibacterium acnes, Propionibacterium freudenreichii, Propionibacterium LG, Propionibacterium, Proteiniborus, Proteiniborus, Proteiniphilum, Proteiniphilum, Proteus, Proteus HS7514, Providencia, Providencia, Pseudobutyrivibrio, Pseudobutyrivibrio bacterium, Pseudobutyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Pseudobutyrivibrio, Pseudochrobactrum, Pseudochrobactrum, Pseudoflavonifractor, Pseudoflavonifractor asf500, Pseudoflavonifractor bacterium, Pseudoflavonifractor capillosus, Pseudoflavonifractor NML, Pseudoflavonifractor, Pseudomonas, Pseudomonas 1043, Pseudomonas 10569, Pseudomonas 127(39-zx), Pseudomonas 12A_19, Pseudomonas 145(38zx), Pseudomonas 22010, Pseudomonas 32010, Pseudomonas 34t20, Pseudomonas 3C_10, Pseudomonas 4-5(2010), Pseudomonas 4-9(2010), Pseudomonas 6-13.J, Pseudomonas 63596, Pseudomonas 82010, Pseudomonas a001-142L, Pseudomonas al01-18-2, Pseudomonas a111-5, Pseudomonas aeruginosa, Pseudomonas agarici, Pseudomonas amspl, Pseudomonas AU2390, Pseudomonas AZ18R1, Pseudomonas azotoformans, Pseudomonas B122, Pseudomonas B65(2012), Pseudomonas bacterium, Pseudomonas BJSX, Pseudomonas BLH-8D5, Pseudomonas BWDY-29, Pseudomonas CA18, Pseudomonas Cantas12, Pseudomonas CB11, Pseudomonas CBZ-4, Pseudomonas cedrina, Pseudomonas CGMCC, Pseudomonas CL16, Pseudomonas CNE, Pseudomonas corrugata, Pseudomonas cuatrocienegasensis, Pseudomonas CYEB-7, Pseudomonas D5, Pseudomonas DAP37, Pseudomonas DB48, Pseudomonas deceptionensis, Pseudomonas Den-05, Pseudomonas DF7EH1, Pseudomonas DhA-91, Pseudomonas DVS14a, Pseudomonas DYJK4-9, Pseudomonas DZQS, Pseudomonas E11_ICE19B, Pseudomonas E2.2, Pseudomonas e2-CDC-TB4D2, Pseudomonas EM189, Pseudomonas enrichment, Pseudomonas extremorientalis, Pseudomonas FA1R/BE/F/GH37, Pseudomonas FA1R/BE/F/GH39, Pseudomonas FA1R/BE/F/GH94, Pseudomonas FLM05-3, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas 'FSL, Pseudomonas G1013, Pseudomonas gingeri, Pseudomonas HC2-2, Pseudomonas HC2-4, Pseudomonas HC2-5, Pseudomonas HC4-8, Pseudomonas HC6-6, Pseudomonas Hg4-06, Pseudomonas HLB8-2, Pseudomonas HLS12-1, Pseudomonas HSF20-13, Pseudomonas HW08, Pseudomonas 11-44, Pseudomonas IpA-92, Pseudomonas IV, Pseudomonas JCM, Pseudomonas jessenii, Pseudomonas JSPBS, Pseudomonas K3R3.1A, Pseudomonas KB40, Pseudomonas KB42, Pseudomonas KB44, Pseudomonas KB63, Pseudomonas KB73, Pseudomonas KK-21-4, Pseudomonas KOPRI, Pseudomonas L1R3.5, Pseudomonas LAB-27, Pseudomonas LAB-44, Pseudomonas Lc10-2, Pseudomonas libanensis, Pseudomonas Ln5C.7, Pseudomonas LS197, Pseudomonas lundensis, Pseudomonas marginalis, Pseudomonas MFY143, Pseudomonas MFY146, Pseudomonas MY1404, Pseudomonas MY1412, Pseudomonas MY1416, Pseudomonas MY1420, Pseudomonas N14zhy, Pseudomonas NBRC, Pseudomonas NCCP-506, Pseudomonas NFU20-14, Pseudomonas NJ-22, Pseudomonas NJ-24, Pseudomonas Nj-3, Pseudomonas Nj-55, Pseudomonas Nj-56, Pseudomonas Nj-59, Pseudomonas Nj-60, Pseudomonas Nj-62, Pseudomonas Nj-70, Pseudomonas NP41, Pseudomonas OCW4, Pseudomonas OW3-15-3-2, Pseudomonas P1(2010), Pseudomonas P2(2010), Pseudomonas P3(2010), Pseudomonas P4(2010), Pseudomonas PD, Pseudomonas PF1B4, Pseudomonas PF2M10, Pseudomonas PILH1, Pseudomonas poae, Pseudomonas proteobacterium, Pseudomonas ps4-12, Pseudomonas ps4-2, Pseudomonas ps4-28, Pseudomonas ps4-34, Pseudomonas ps4-4, Pseudomonas psychrophila, Pseudomonas putida, Pseudomonas R-35721, Pseudomonas R-37257, Pseudomonas R-37265, Pseudomonas R-37908, Pseudomonas RBE1CD-48, Pseudomonas RBE2CD-42, Pseudomonas regd9, Pseudomonas RKS7-3, Pseudomonas S2, Pseudomonas seawater, Pseudomonas SGb08, Pseudomonas SGb120, Pseudomonas SGb396, Pseudomonas sgn, Pseudomonas 'Shk, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas taetrolens, Pseudomonas tolaasii, Pseudomonas trivialis, Pseudomonas TUT1023, Pseudomonas, Pseudomonas W15Feb26, Pseudomonas W15Feb4, Pseudomonas W15Feb6, Pseudomonas WD-3, Pseudomonas WR4-13, Pseudomonas WR7#2, Pseudomonas Y1000, Pseudomonas ZS29-8, Psychrobacter, Psychrobacter umb13d, Psychrobacter, Pyramidobacter, Pyramidobacter piscolens, Pyramidobacter, Rahnella, Rahnella aquatilis, Rahnella carotovorum, Rahnella GIST-WP4w1, Rahnella LR113, Rahnella, Rahnella Z2-S1, Ralstonia, Ralstonia bacterium, Ralstonia, Raoultella, Raoultella B19, Raoultella enrichment, Raoultella planticola, Raoultella sv6xvii, Raoultella SZ015, Raoultella, Renibacterium, Renibacterium G20, Rhizobium, Rhizobium leguminosarum, Rhodococcus, Rhodococcus erythropolis, Rhodopirellula, Rhodopirellula, Riemerella, Riemerella anatipestifer, Rikenella, Rikenella, Robinsoniella, Robinsoniella peoriensis, Robinsoniella, Roseburia, Roseburia 11SE37, Roseburia bacterium, Roseburia cecicola, Roseburia DJF VR77, Roseburia faecis, Roseburia fibrisolvens, Roseburia hominis, Roseburia intestinalis, Roseburia inulinivorans, Roseburia, Roseibacillus, Roseibacillus, Rothia, Rothia, Rubritalea, Rubritalea, Ruminococcus, Ruminococcus 25F6, Ruminococcus albus, Ruminococcus bacterium, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus champanellensis, Ruminococcus DJF VR87, Ruminococcus flavefaciens, Ruminococcus gauvreauii, Ruminococcus lactaris, Ruminococcus NK3A76, Ruminococcus, Ruminococcus YE71, Saccharofermentans, Saccharofermentans, Salinicoccus, Salinicoccus, Salinimicrobium, Salinimicrobium, Salmonella, Salmonella agglomerans, Salmonella bacterium, Salmonella enterica, Salmonella freundii, Salmonella hermannii, Salmonella paratyphi, Salmonella SL0604, Salmonella subterranea, Salmonella, Scardovia, Scardovia oral, Schwartzia, Schwartzia, Sedimenticola, Sedimenticola, Sediminibacter, Sediminibacter, Selenomonas, Selenomonas fecal, Selenomonas, Serpens, Serpens, Serratia, Serratia 1135, Serratia 136-2, Serratia 5.1R, Serratia AC-CS-1B, Serratia AC-CS-B2, Serratia aquatilis, Serratia bacterium, Serratia B S26, Serratia carotovorum, Serratia DAP6, Serratia enrichment, Serratia F2, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia J145, Serratia JM983, Serratia liquefaciens, Serratia marcescens, Serratia plymuthica, Serratia proteamaculans, Serratia proteolyticus, Serratia ptz-16s, Serratia quinivorans, Serratia SBS, Serratia SS22, Serratia trout, Serratia UA-G004, Serratia, Serratia White, Serratia yellow, Shewanella, Shewanella baltica, Shewanella, Slackia, Slackia intestinal, Slackia isoflavoniconvertens, Slackia NATTS, Slackia, Solibacillus, Solibacillus, Solobacterium, Solobacterium moorei, Solobacterium, Spartobacteria_genera_incertae_sedis, Spartobacteria_genera_incertae_sedis, Sphingobium, Sphingobium, Sphingomonas, Sphingomonas, Sporacetigenium, Sporacetigenium, Sporobacter, Sporobacter, Sporobacterium, Sporobacterium olearium, Staphylococcus, Staphylococcus epidermidis, Staphylococcus PCA17, Staphylococcus, Stenotrophomonas, Stenotrophomonas, Streptococcus, Streptococcus 1606-02B, Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus bacterium, Streptococcus bovis, Streptococcus ChDC, Streptococcus constellatus, Streptococcus CR-314S, Streptococcus criceti, Streptococcus cristatus, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus enrichment, Streptococcus equi, Streptococcus equinus, Streptococcus ES11, Streptococcus eubacterium, Streptococcus fecal, Streptococcus gallinaceus, Streptococcus gallolyticus, Streptococcus gastrococcus, Streptococcus genomosp, Streptococcus gordonii, Streptococcus I5, Streptococcus infantarius, Streptococcus intermedius, Streptococcus Je2, Streptococcus JS-CD2, Streptococcus LRC, Streptococcus luteciae, Streptococcus lutetiensis, Streptococcus M09-11185, Streptococcus mitis, Streptococcus mutans, Streptococcus NA, Streptococcus nlaezlc353, Streptococcus nlaezlp68, Streptococcus nlaezlp758, Streptococcus nlaezlp807, Streptococcus oral, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus phocae, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pyogenes, Streptococcus S16-08, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus symbiont, Streptococcus thermophilus, Streptococcus TW1, Streptococcus, Streptococcus vestibularis, Streptococcus warneri, Streptococcus XJ-RY-3, Streptomyces, Streptomyces malaysiensis, Streptomyces MVCS6, Streptophyta, Streptophyta cordifolium, Streptophyta ginseng, Streptophyta hirsutum, Streptophyta oleracea, Streptophyta sativa, Streptophyta sativum, Streptophyta sativus, Streptophyta tabacum, Streptophyta, Subdivision3_genera_incertae_sedis, Subdivision3_genera_incertae_sedis, Subdoligranulum, Subdoligranulum bacterium, Subdoligranulum ic1393, Subdoligranulum ic1395, Subdoligranulum, Subdoligranulum variabile, Succiniclasticum, Succiniclasticum, Sulfuricella, Sulfuricella, Sulfurospirillum, Sulfurospirillum, Sutterella, Sutterella, Sutterella wadsworthensis, Syntrophococcus, Syntrophococcus, Syntrophomonas, Syntrophomonas bryantii, Syntrophomonas, Syntrophus, Syntrophus, Tannerella, Tannerella, Tatumella, Tatumella, Thermofilum, Thermofilum, Thermogymnomonas, Thermogymnomonas, Thermovirga, Thermovirga, Thiomonas, Thiomonas ML1-46, Thorsellia, Thorsellia carsonella, TM7_genera_incertae_sedis, TM7_genera_incertae_sedis, Trichococcus, Trichococcus, Turicibacter, Turicibacter sanguinis, Turicibacter, Vagococcus, Vagococcus bfs11-15, Vagococcus, Vampirovibrio, Vampirovibrio, Varibaculum, Varibaculum, Variovorax, Variovorax KS2D-23, Veillonella, Veillonella dispar, Veillonella MSA12, Veillonella OK8, Veillonella oral, Veillonella parvula, Veillonella tobetsuensis, Veillonella, Vibrio, Vibrio 3C1, Vibrio, Victivallis, Victivallis, Victivallis vadensis, Vitellibacter, Vitellibacter, Wandonia, Wandonia haliotis, Weissella, Weissella cibaria, Weissella confusa, Weissella oryzae, Weissella, Yersinia, Yersinia 9gw38, Yersinia A125, Yersinia aldovae, Yersinia aleksiciae, Yersinia b702011, Yersinia bacterium, Yersinia bercovieri, Yersinia enterocolitica, Yersinia entomophaga, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia MAC, Yersinia massiliensis, Yersinia mollaretii, Yersinia nurmii, Yersinia pekkanenii, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia rohdei, Yersinia ruckeri, Yersinia s10fe31, Yersinia s17fe31, Yersinia s4fe31, Yersinia, Yersinia YEM17B.

Accuracy and Sensitivity

[0145] The methods provided herein can provide strain classification of a genera, species or sub-strain level of one or more microbes in a sample with an accuracy of greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%. The methods provided herein can provide strain quantification of a genera, species or sub-strain level of one or more microbes in a sample with an accuracy of greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%.

[0146] In some applications a similar microbiome profile from a patient to a reference profile indicates an increased likelihood of one or more of: a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management. In some applications, a dissimilar microbiome profile from a patient to a reference profile indicates one or more of: an increased likelihood of a poor clinical outcome, good clinical outcome, high risk of disease, low risk of disease, complete response, partial response, stable disease, non-response, and recommended treatments for disease management.

[0147] The methods provided herein can provide a health status of a subject with a specificity greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%, or 99.9% ROC. The methods provided herein can provide a health status of a subject with sensitivity greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%, or 99.9% ROC.

[0148] The diagnostic methods provided by the present disclosure for the diseases provided herein can have at least one of a sensitivity of 70% or greater and specificity of greater than 70% based on measurement of 15 or fewer microbes in the biological sample. Such diagnostic method can have at least one of a sensitivity greater than 70% and specificity greater than 70% based on measurement of no more than 2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, or 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbes, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes or 800 or fewer microbes.

[0149] The microbial profile for a subject provided by the present disclosure can have an accuracy of 70% or greater based on measurement of 15 or fewer microbes in the biological sample. Such profiling method can have at least an accuracy greater than 70% based on measurement of no more than 2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewer microbes, or 100 or fewer microbes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewer microbes, 500 or fewer microbes, 600 or fewer microbes, 700 or fewer microbes or 800 or fewer microbes.

III. Computer Systems

[0150] The current disclosure provides computer systems for implementing any of the methods described herein. A computer system may be used to implement one or more steps including, sample collection, sample processing, detecting, quantifying one or more microbes, generating a profile data, comparing said data to a reference, generating a subject-specific microbiome profile, comparing the subject-specific profile to a reference profile, receiving medical history, receiving medical records, receiving and storing data obtained by one or more methods described herein, analyzing said data, generating a report, and reporting results to a receiver.

[0151] For example, provided herein are computer systems for detecting a presence or absence of a microbes. Also provided herein are computer systems for detecting a presence or absence of bacteria, fungi, archaea or other elements that comprise and maintain a microbiome.

[0152] Computer systems described herein may comprise computer-executable code for performing any of the algorithms described herein. Computer systems described herein may comprise computer-executable code for performing any of the algorithms and using the database as herein.

[0153] FIG. 1 depicts an exemplary computer system 100 adapted to implement a method described herein. The system 100 includes a central computer server 101 that is programmed to implement exemplary methods described herein. The server 101 includes a central processing unit (CPU, also "processor") 105 which can be a single core processor, a multi core processor, or plurality of processors for parallel processing. The server 101 also includes memory 110 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 115 (e.g. hard disk); communications interface 120 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 125 which may include cache, other memory, data storage, and/or electronic display adaptors. The memory 110, storage unit 115, interface 120, and peripheral devices 125 are in communication with the processor 105 through a communications bus (solid lines), such as a motherboard. The storage unit 115 can be a data storage unit for storing data. The server 101 is operatively coupled to a computer network ("network") 130 with the aid of the communications interface 120. The network 130 can be the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a telecommunication or data network. The network 130 in some cases, with the aid of the server 101, can implement a peer-to-peer network, which may enable devices coupled to the server 101 to behave as a client or a server.

[0154] The storage unit 115 can store files, such as subject reports, and/or communications with the caregiver, sequencing data, data about individuals, or any aspect of data associated with the invention.

[0155] The server can communicate with one or more remote computer systems through the network 130. The one or more remote computer systems may be, for example, personal computers, laptops, tablets, telephones, Smart phones, or personal digital assistants.

[0156] In some applications the computer system 100 includes a single server 101. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the internet.

[0157] The server 101 can be adapted to store measurement data or a database as provided herein, patient information from the subject, such as, for example, polymorphisms, mutations, medical history, family history, demographic data and/or other clinical or personal information of potential relevance to a particular application. Such information can be stored on the storage unit 115 or the server 101 and such data can be transmitted through a network.

[0158] Methods as described herein can be implemented by way of machine (or computer processor) executable code (or software) stored on an electronic storage location of the server 101, such as, for example, on the memory 110, or electronic storage unit 115. During use, the code can be executed by the processor 105. In some cases, the code can be retrieved from the storage unit 115 and stored on the memory 110 for ready access by the processor 105. In some situations, the electronic storage unit 115 can be precluded, and machine-executable instructions are stored on memory 110. Alternatively, the code can be executed on a second computer system 140.

[0159] Aspects of the systems and methods provided herein, such as the server 101, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless likes, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" can refer to any medium that participates in providing instructions to a processor for execution

[0160] Computer systems described herein may comprise computer-executable code for performing any of the algorithms or algorithms-based methods described herein. In some applications the algorithms described herein will make use of a memory unit that is comprised of at least one database.

[0161] Data relating to the present disclosure can be transmitted over a network or connections for reception and/or review by a receiver. The receiver can be but is not limited to the subject to whom the report pertains; or to a caregiver thereof, e.g., a health care provider, manager, other health care professional, or other caretaker; a person or entity that performed and/or ordered the analysis. The receiver can also be a local or remote system for storing such reports (e.g. servers or other systems of a "cloud computing" architecture). In one embodiment, a computer-readable medium includes a medium suitable for transmission of a result of an analysis of a biological sample using the methods described herein.

[0162] A. Databases

[0163] Computer systems disclosed herein may comprise a memory unit. The memory unit can be configured to receive data comprising extracting data from a pubic database, detecting, quantifying and profiling one or more microbiomes. The microbiome profile can be any organism known to comprise a microbiome. Examples of such organisms are provided herein.

[0164] There are several public microbe (bacteria, fungi, and archaea) and viral protein and genome databases known in the art. The present methods of the disclosure can be used with such public databases. Examples of public databases include but are not limited to Biocyc, Ensembl Bacteria, The Integrated Microbial Genomes, MicrobesOnline, Microbial Genomes from Genome Channel, Microbial Genomes at NCBI, RCSB protein database, Sanger Centre Bacterial Genomes, Ribosomal Database Project (RDP), or DOE JGI Microbial Genomics Database.

[0165] The current disclosure also provides for a database that has additional or more accurate microbe information such as the composition of particular microbiomes in a particular cohort, or microbiome reference profiles of a particular cohort. Such database can include but are not limited to additional or more accurate sequences comprising the 16S subunit of ribosome for a given microbe strain, additional or more accurate sequence comprising the 23S subunit of ribosome for a given microbe strain, additional or more accurate information of the sequence comprising the intergenic region between the 16S subunit and 23S subunit of ribosome, additional or more accurate information of the sequence comprising variable regions in the 16S ribosome for a particular strain, additional or more accurate information of the sequence comprising variable regions in the 23S subunit of ribosome for a particular strain, additional or more accurate information of the sequence comprising variable regions with a high accuracy in strain resolution at the genus level, additional or more accurate information of the sequence comprising variable regions with a high accuracy in strain resolution at the species level, or additional or more accurate information of the sequence comprising variable regions with a high accuracy in strain resolution at the sub-type level.

[0166] Such a database that has additional or more accurate genome information can be comprised of sequence reads greater than 500 base pair, 600 base pair, 700 base pair, 800 base pair, 900 base pair, 1000 base pair, 1100 base pair, 1200 base pair, 1300 base pair, 1400 base pair, 16S subunit of ribosome for a given microbe strain. Such a database can be comprised of sequence reads greater than 500 base pair, 600 base pair, 700 base pair, 800 base pair, 900 base pair, 1000 base pair, 1100 base pair, 1200 base pair, 1300 base pair, 1400 base pair, 16S or 23S subunit of ribosome for a given microbe strain.

[0167] Such a database can be comprised of sequence reads greater than 500 base pair, 600 base pair, 700 base pair, 800 base pair, 900 base pair, 1000 base pair, 1100 base pair, 1200 base pair, 1300 base pair, 1400 base pair, comprising the intergenic region between the 16S subunit and 23S subunit of ribosome for a given bacterial strain.

[0168] Such a database can be comprised of sequence reads greater than 500 base pair, 600 base pair, 700 base pair, 800 base pair, 900 base pair, 1000 base pair, 1100 base pair, 1200 base pair, 1300 base pair, 1400 base pair, 1500 base pair comprising the variable regions in the 16S ribosome. Such a database can be comprised of sequence reads greater than 500 base pair, 600 base pair, 700 base pair, 800 base pair, 900 base pair, 1000 base pair, 1100 base pair, 1200 base pair, 1300 base pair, 1400 base pair, 1500 base pair comprising the variable regions in the 16S or 23S ribosome.

[0169] Such a database can further comprise of additional or more accurate proteome information can be comprised of sequence reads greater than 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids, 1400 amino acids, 16S subunit of ribosome for a given microbe strain. Such a database can be comprised of sequence reads greater than 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids, 1400 amino acids, 16S or 23S subunit of ribosome for a given microbe strain.

[0170] Such a database can be comprised of sequence reads greater than 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids, 1400 amino acids, comprising the intergenic region between the 16S subunit and 23S subunit of ribosome for a given bacterial strain.

[0171] Such a database can be comprised of sequence reads greater than 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids, 1400 amino acids, 1500 amino acids comprising the variable regions in the 16S ribosome.

[0172] Such a database can be comprised of sequence reads greater than 500 amino acids, 600 amino acids, 700 amino acids, 800 amino acids, 900 amino acids, 1000 amino acids, 1100 amino acids, 1200 amino acids, 1300 amino acids, 1400 amino acids, 1500 amino acids comprising the variable regions in the 16S or 23S ribosome.

[0173] The database maybe located on central server containing the computer-executable code that allows access to a user. The user can connect to the central server through a physical connection or cloud-based connection depending on the application. In some applications a portion of the database and necessary executable code will be supplied to as user on appropriate storage media.

[0174] B. Computer Generated Report

[0175] The computer system can further comprise computer-executable code for providing a report communicating the detecting, measuring, or determining a profile of a microbiome from a subject. Measuring, or determining a profile of a microbiome can include the use of a database as provided herein.

[0176] Computer systems disclosed herein may comprise computer-executable code for performing at least one of: generating a cohort-generalized microbiome profile or a subject-specific microbiome profile based upon the measurement data from a biological sample from the subject, comparing the cohort-generalized microbiome profile or subject-specific microbiome profile to at least one reference and determining the health status of a subject.

[0177] In some applications the computer system can access the computer-executable code by having a connection to a central server that contained the computer-executable code to generate a report comprising at least one clinical recommendation such as for example, disease state, diagnosis, prognosis, treatment suggestions or procedures for clinical management in a subject which can be retrieved by a health worker or clinician via said central server. The connection to a central server containing the computer-executable code can be a physical connection or cloud-based connection depending on the application.

IV. Kits

[0178] The disclosure provides kits. A kit described herein can comprise one or more compositions, reagents, buffers, components for measuring or detecting one or more microbes or microbiomal profiles by a method provided herein. A kit as can further comprise instructions for practicing any of the methods provided herein. For example, instructions can include specifics sample preparation steps for the biological samples as provided herein and it for measuring or detecting. Likewise, the contents of the kit will be tailored to its particular application and sample type.

[0179] The kits can further comprise reagents to enable the detection by such applications as PCR, DNA/RNA array, protein array, sequencing, mass spectrometry, immunohistochemistry, laser cell microdissection, high-content cell screening, flow cytometry, which are suitable with the methods described herein for detection and determination of a subject's prognosis, prediction of response, and diagnosis.

[0180] Kits can further comprise a software package for measuring or determining of a microbiome profile as, which as described herein, can include reference microbiome profiles or other health related data. In some applications the kits software package including connection to a central server to conduct for measuring or determining and can generate a report comprising at least one clinical recommendation such as for example, disease state, diagnosis, prognosis, treatment suggestions or procedures for clinical management in a subject which can be retrieved by a health worker or clinician via said central server.

[0181] In some applications, kits can further comprise a report. The report can be a paper or an electronic report. The report can be generated by computer software (e.g. computer-executable code) provided with the kit, or by a computer sever which the user uploads to a website wherein the computer server generates the report.

[0182] In some applications the kit can provide for profiling more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 microbes at the genera, species, sub-strain level or a combination thereof.

[0183] In general, kits will comprise of a packaging material. As used herein, the term "packaging material" can refer to a physical structure housing the components of the kit. The packaging material can maintain sterility of the kit components, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, etc.). Depending on the applications, kits can also include such materials as a buffering agent, a preservative, or a protein or nucleic acid stabilizing agents or any such materials that are necessary for the stable transport of biological samples and reagents.

[0184] V. Microbial-Based Therapeutics and Cosmetics

[0185] Provided herein are compositions that may be administered as therapeutics or cosmetics or as a cosmetic. One or more, or combination thereof the microorganisums provided herein can be used to created various formulations for treating a subject. The present disclosure provides therapeutic or cosmetic formulations for the following conditions IBD, preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type 2 diabetes, C. difficile, IBS, asthma, autism, psoriasis, allergies, cardiovascular disease, cancer, depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, and type 1 diabetes.

[0186] A. Formulations

[0187] Formulations provided herein can include the addition of one or more genetic elements to the therapeutics or cosmetics in order to enchance stability. Formulations provided herein can include those suitable for oral including buccal and sub-lingual, rectal, intranasal, topical, transdermal, transdermal patch, pulmonary, vaginal, suppository, or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or in a form suitable for administration by aerosolization, inhalation or insufflation. General information on drug delivery systems can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999).

[0188] The appropriate quantity of a therapeutic or cosmetic composition to be administered, the number of treatments, and unit dose will vary according to the subject and the disease state of the subject. The person responsible for administration will determine the appropriate dose, number of treatments, etc. for the subject.

[0189] In various applications, the therapeutic or cosmetic composition can include carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0190] In some applications, the therapeutic or cosmetic composition is substantially free of preservatives. In other applications, the composition may contain at least one preservative. General methodology on pharmaceutical dosage forms is found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999). It will be recognized that, while any suitable carrier known to those of ordinary skill in the art may be employed to administer the compositions provided herein, the type of carrier will vary depending on the mode of administration. A thorough discussion of acceptable carriers/excipients can be found in Remington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition, 2000: Williams and Wilkins PA, USA.

[0191] A therapeutic or cosmetic composition may also be encapsulated within liposomes using well-known technology. Biodegradable microspheres may also be employed as carriers for the therapeutics or cosmetics compositions provided herein. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

[0192] The composition may be administered in liposomes or microspheres or microparticles. Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. U.S. Pat. No. 4,789,734, the contents of which are hereby incorporated by reference, describes methods for encapsulating biological materials in liposomes. A review of known methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, pp. 2.sup.87-341 (Academic Press, 1979).

[0193] Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents of which are hereby incorporated by reference.

[0194] The compositions provided herein may be formulated as a sterile solution or suspension, in suitable vehicles, well known in the art. The therapeutic or cosmetic compositions may be sterilized by conventional techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. Suitable formulations and additional carriers are described in Remington "The Science and Practice of Pharmacy" (20th Ed., Lippincott Williams & Wilkins, Baltimore Md.), the teachings of which are incorporated by reference in their entirety herein.

VI. Indications

[0195] As mentioned above, research indicates that microbiomes can have an effect on a subject's disease status and clinical treatment response. For example, obese and lean individuals can be categorized based on differences in specific species of microbes (Future Microbiol. (2012) 7(1): 91-109. Additionally, in another example the administration of Lactobacillus gasseri SBT2055 to lean individuals had observable weight loss (Micr. Path. (2012) 53(2): 100-108; Eur J Clin Nutr (2010) 64:636-43). On the other hand for obese individuals it was Lactobacillus plantarum that had the biggest weight loss effect (Micr. Path. (2012) 53(2): 100-108; Eur J Clin Nutr (2010) 64:636-43). These studies indicate that specifically tailoring treatment to a subjects' microbiome may have beneficial effects on a subject's health status or ameliorate certain symptoms associated with the onset of an indication.

[0196] Furthermore, in non-obese individuals, the acetate producing fermentation reaction is limited by the partial pressure of hydrogen which creates a thermodynamic roadblock to further fermentation (Dolfing & Tiedje, 54 Appl. Environ. Microbiol. 1871-3 1988; Schink, 61 Microbiol. Mol. Bio. Rev. 262-80 1997). Obese individuals on the other hand have hydrogen-oxidizing methanogens that can consume the hydrogen and thus allow the acetate producing fermentation reaction to continue. The gut of obese individuals actually uniquely contains hydrogen-oxidizing methanogenic Archaea (Zhang et al PNAS 106:2365-70 2009) and higher levels of Prevotellaceae (Bacteroidetes phylum) (Turnbaugh 2006), which are known to perform the carbohydrate fermentation reaction to acetate and hydrogen.

[0197] Indications that can be used with the present disclosure include, but are not limited to the following: IBD, preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type 2 diabetes, C. difficile, IBS, asthma, autism, psoriasis, allergies, cardiovascular disease, cancer, depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, and type 1 diabetes.

[0198] Inflammatory bowel disease (IBD) involves chronic inflammation of all or part of the digestive tract. IBD primarily includes ulcerative colitis and Crohn's disease. IBD can be painful and debilitating, and sometimes leads to life-threatening complications. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for IBD, ulcerative colitis or Crohn's disease. The present disclosure also provides therapeutic or cosmetic formulations for treatment of inflammatory bowel disease conditions.

[0199] Preterm labor occurs when contractions begin to open the cervix before 37 weeks of pregnancy. The earlier premature birth happens, the greater the health risks for the developing baby. Many premature babies need special care in the neonatal intensive care unit. Premature babies can also have long-term mental and physical disabilities. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for preterm labor. The present disclosure also provides therapeutic or cosmetic formulations for treatment of preterm labor.

[0200] Obesity is a complex disorder involving an excessive amount of body fat. Obesity increases the risk of diseases and health problems such as heart disease, diabetes and high blood pressure. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for obesity. The present disclosure also provides therapeutic or cosmetic formulations for treatment of obesity conditions.

[0201] Peripheral neuropathy is the most common form of diabetic neuropathy. The feet and legs are often affected first, followed by the hands and arms. Possible signs and symptoms of peripheral neuropathy can include serious foot problems, such as ulcers, infections, deformities, and bone and joint pain. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for diabetic neuropathy. The present disclosure also provides therapeutic or cosmetic formulations for treatment of diabetic neuropathy conditions.

[0202] Bacteremia or septicemia refers to the presence of bacteria in the blood. A diagnosis of bacteremia is usually confirmed by a blood culture. Treatment usually requires hospitalization and intravenous antibiotics. Without prompt treatment, bacteremia can quickly progress to severe sepsis. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for bacteremia, which can include the antibiotic susceptibilities of the infection. The present disclosure also provides therapeutic or cosmetic formulations for treatment of bacteremia.

[0203] Acne is a skin condition that occurs when the hair follicles become plugged with oil and dead skin cells. Acne most commonly appears on the face, neck, chest, back and shoulders. Depending on its severity of the acne, this condition can cause emotional distress and lead to scarring of the skin. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of that includes a report that gives guidance on health status or treatment modalities for acne. The present disclosure also provides therapeutic or cosmetic formulations for treatment of acne conditions.

[0204] Infantile colic physicians and parents use the term colic to describe an infant with excessive crying, irritability, or fussiness. Babies with colic often cry more than three hours a day, three days a week for three weeks or longer. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for infantile colic. The present disclosure also provides therapeutic or cosmetic formulations for treatment of the above-mentioned condition.

[0205] Type 2 diabetes, once known as adult-onset or noninsulin-dependent diabetes, is a chronic condition that affects the way the body metabolizes glucose. With type 2 diabetes, the body either resists the effects of insulin or doesn't produce enough insulin to maintain a normal glucose level. Untreated, type 2 diabetes can be life-threatening. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for type 2 diabetes. The present disclosure also provides therapeutic or cosmetic formulations for treatment of type 2 diabetes.

[0206] Clostridium difficile, often called "C. difficile" or "C. diff", is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for infections such as C. difficile. The present disclosure also provides therapeutic or cosmetic formulations for treatment of Clostridium difficile infections.

[0207] Asthma is a condition in which the airways narrow and swell and produce extra mucus. This can make breathing difficult and trigger coughing, wheezing and shortness of breath. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for asthma. The present disclosure also provides therapeutic or cosmetic formulations for treatment of asthma.

[0208] Autism spectrum disorder is a serious neurodevelopmental disorder that impairs a child's ability to communicate and interact with others. It also includes restricted repetitive behaviors, interests and activities. Autism spectrum disorder (ASD) is now defined by the American Psychiatric Association's Diagnosis and Statistical Manual of Mental Disorders (DSM-5) as a single disorder that includes disorders that were previously considered separate--autism, Asperger's syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for autism spectrum disorder. The present disclosure also provides therapeutic or cosmetic formulations for treatment of autism spectrum disorders.

[0209] Psoriasis is a common, persistent, long-lasting (chronic) skin condition that changes the life cycle of skin cells. Psoriasis causes cells to build up rapidly on the surface of the skin. The extra skin cells form thick, silvery scales and itchy, dry, red patches that are sometimes painful. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for Psoriasis. The present disclosure also provides therapeutic or cosmetic formulations for treatment of psoriasis or a similar skin condition.

[0210] Allergies occur when the immune system reacts to a foreign substance such as pollen, bee venom or pet dander. When you come into contact with the allergen, the immune system's reaction can inflame the skin, sinuses, airways or digestive system. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for allergies. The present disclosure also provides therapeutic or cosmetic formulations for treatment of allergies.

[0211] Cardiovascular diseases can affect the heart, arteries and veins of the body. Examples of some cardiovascular disease include but are not limited to heart valve disease, coronary artery disease, congenital heart disease in adults and congenital heart spontaneous coronary artery dissection, heart failure, heart rhythm disorders (arrhythmias). The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for cardiovascular disease. The present disclosure also provides therapeutic or cosmetic formulations for treatment of the above-mentioned cardiovascular conditions.

[0212] Cancer refers to any one of a large number of proliferative diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal body tissues and organs. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for cancer or other proliferative diseases. The present disclosure also provides therapeutic or cosmetic formulations for treatment of cancer.

[0213] Depression also called major depression, major depressive disorder or clinical depression is a mood disorder that causes a persistent feeling of sadness and loss of interest. It can affect how a person feels, thinks and behaves and can lead to a variety of emotional and physical problems. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for depression disorders. The present disclosure also provides therapeutic or cosmetic formulations for treatment of the above-mentioned depression conditions.

[0214] Cystic fibrosis is a life-threatening genetic disorder that causes severe damage to the lungs and digestive system. Cystic fibrosis affects the cells that produce secreted fluids such as mucus, sweat and digestive juices that act as lubricants in the body. These secreted fluids are normally thin and slippery but in cystic fibrosis the secretions to become thick and sticky resulting in plugging up tubes, ducts and passageways, especially in the lungs and pancreas. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for cystic fibrosis. The present disclosure also provides therapeutic or cosmetic formulations for treatment of cystic fibrosis.

[0215] Multiple sclerosis is a disease in which the immune system attacks the protective sheath (myelin) that covers the nerves. Myelin damage disrupts communication between the brain and the rest of the body. Ultimately, the nerves themselves may deteriorate a process that's currently irreversible. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for multiple sclerosis. The present disclosure also provides therapeutic or cosmetic formulations for treatment of multiple sclerosis.

[0216] Urinary tract infection is an infection in any part of the urinary system (e.g. kidneys, ureters, bladder and urethra). Most infections involve the lower urinary tract--the bladder and the urethra. Infection limited to the bladder can be painful and annoying. However, serious consequences can occur if a UTI spreads to the kidneys. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for urinary tract infections. The present disclosure also provides therapeutic or cosmetic formulations for treatment of infections in any part of the urinary system.

[0217] Radiation enteropathy is radiation-induced GI injuries of the colon and rectum to the small bowel. Radiotherapy is a mainstay of oncological treatment for a variety of malignant diseases and is commonly administered to the abdomen and pelvis of patients with gastrointestinal (GI), urological and gynaecological cancers. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for radiation enteropathy. The present disclosure also provides therapeutic or cosmetic formulations for treatment of radiation-induced injury conditions.

[0218] Drug metabolism refers to the rate at which the body breaks down as drug after administration. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for drug metabolism in a patient. The present disclosure also provides therapeutic or cosmetic formulations for treatment of drug metabolism conditions.

[0219] Chronic fatigue syndrome is a complicated disorder characterized by extreme fatigue that can't be explained by any underlying medical condition. The fatigue may worsen with physical or mental activity, but doesn't improve with rest. The cause of chronic fatigue syndrome is currently unknown. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for chronic fatigue syndrome. The present disclosure also provides therapeutic or cosmetic formulations for treatment of CFS conditions.

[0220] Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin, a hormone needed to allow sugar (glucose) to enter cells to produce energy. Various factors may contribute to type 1 diabetes, including genetics and exposure to certain viruses. Although type 1 diabetes typically appears during childhood or adolescence, it also can develop in adults. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for type 1 diabetes. The present disclosure also provides therapeutic or cosmetic formulations for treatment of type 1 diabetes.

[0221] Dental cavities are caused by the conversion of sugar from food to elongated, sticky sugar chains through a bacterially produced glucansucrase enzyme. Attempts to directly inhibit this enzyme have failed because it is evolutionarily closely related to amylase, which is the enzyme used to break down starch. A more effective approach would be to reduce the proportion of Streptococcus mutans, which is the bacteria associated with tooth decay. This would leave intact the useful enzymes used by the body to break down starches while simultaneously minimizing the rate of cavity formation. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for tooth decay. The present disclosure also provides provides therapeutic or cosmetic formulations for treatment of tooth decay conditions.

[0222] Halitosis is a dental condition in which excessively bad breath is produced by the microbial flora present in an individual's mouth. It is known that the most problematic types are the gram-negative bacteria (e.g. Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola). By using the methods outlined in this disclosure, a strain level list of the most problematic bacteria, as well as the most protective, will enable a directed therapeutic/cosmetic formulation to treat halitosis. The methods, compositions, systems and kits of the present disclosure provide for a diagnostic assay of at least one microbiome that includes a report that gives guidance on health status or treatment modalities for halitosis. The present disclosure also provides therapeutic or cosmetic formulations for treatment of halitosis.

VII. Examples

Example 1: Comparison of Microbe Classification Accuracy

[0223] As more bacterial strains are identified, it has become clear that just sequencing a few of the hypervariable regions does not provide enough distinction. There are classes of bacteria now characterized that share homologous hypervariable regions. Therefore, the need to sequence the entire 16S and/or 23S and include all of the hypervariable regions is necessary to make accurate classification calls. Comparison simulations experiments were conducted with the methods of the present disclosure and the current methods in the field to determine if the methods of the present disclosure provide increased accuracy for microbe classification.

[0224] Using long read length sequence coverage of the 16S and/or 23S ribosomal subunits, allows for each sequence read yield an unambiguous assignment of bacterial identification. The current shorter read length platforms, covers only 1-3 hypervariable regions FIG. 2A. These shorter read length platforms are currently performing at, or below, a 60-80% classification accuracy. A read length that can span the entirety of the 16S operon will perform at a 90-99% classification accuracy FIG. 2B. The plot shown in FIG. 2B presents classification accuracy based upon sequencing shorter hyper-variable regions (lower lines) vs. sequencing the entire 16S subunit in a single sequence read (upper line). The plots are produced using calculations derived from data from the Ribosomal Database Project. Moreover, even accounting for higher error rates in certain longer read sequencing technologies still yields substantially better classification accuracy. By extension, sequencing the entirety of the ribosomal RNA operon (rRNA that includes--16S, intergenic, and 23 S regions) would increase the classification capability of the method.

Example 2: Affect of Diet on a Subject's Gut Microbiome and Obesity (Prophetic Example)

[0225] Using the methods provided herein, experiments were conducted to determine if a particular diet regimen can changes a subjects' gut microbiome profile. FIG. 3 depicts changes measured and detected in an individual's gut microbiome profile to changes in diet using the methods described herein. Weight (dashed line) is a lagging indicator, when compared to the change in the microbiome structure.

[0226] A report containing individualized dieting recommendation was generated based on quantitative microbiome profile using the computer system shown in FIG. 1 was provided to a healthcare professional.

Example 3: Comparison of Microbe Resolution at Strain Level of a Microbiome

[0227] Comparison simulation experiments were conducted with the methods of the present disclosure to determine if the methods of the present disclosure provide increased microbe classification accuracy at the strain level compared to the current methods being used in the field.

[0228] Samples were collected in the form of skin swabs. Briefly, a sterile swab is first dipped into a tube containing sterile 1.times.PBS to wet; the swab was then swiped across the area of interest 10-20 times; next the swab was gently dipped into 300 uL of lysis buffer (described herein) in a sterile 1.5 mL tube; and the swab is left in the microcentrifuge tube until nucleic acid extraction. The subsequent extraction of DNA from human skin microbiome samples includes the removal of the exonuclease was conducted followed by adapter ligation at various higher concentrations. Next, size-selection using Ampure and Blue Pippen approaches were performed to enrich for the expected length amplicon species. After extraction, the nucleic acid samples were selected for the proper size. Next, PCR amplification reactions were conducted to prepare the libraries for sequencing. Forward and reverse primers were selected based on empirical data that indicate which sets had minimal self-complimentarity. Finally samples were sequenced using long read length sequence technology.

[0229] FIG. 4A depicts strain level resolution data with the methods provided for Nitrosomonas genus, percentage strain classification accuracy is shown in the Y-axis. FIG. 4B depicts strain level resolution data with the methods provided for Staphylococcus genus, percentage strain classification accuracy is shown in the Y-axis. FIG. 6A standard resolution of a microbiome using current technology FIG. 6B resolution of a microbiome using a method described herein.

Example 4: Study of Microbial-Based Therapeutic Application on Acne (Prophetic Example)

[0230] Experiments using the methods provided herein were conducted to determine if the application of Nitrosomonas to the skin can have beneficial effects on a person suffering from acne.

[0231] Samples were prepared for skin samples using the following steps a sterile swab is first dipped into a tube containing sterile 1.times.PBS to wet; the swab is swiped across the area of interest 10-20 times with enough vigor that the skin is slightly pink/red colored afterwards; the swab is gently dipped into 300 uL of Lysis Buffer (described below) in a sterile 1.5 mL tube; the swab is left in the microcentrifuge tube for shipping and then processed for nucleic acid extraction and long read sequence analysis using the methods provided herein.

[0232] FIGS. 5A and 5B depict expected results from the study. FIG. 5A depicts how the methods of this disclosure can be used to quantify the successful application of the microbial therapeutic/cosmetic over time. The addition of a non-commensal strain to the consortia, aids in distinguishing applied strains from the background variation of individual microbiomes for that site (e.g. skin, gut, mouth, etc.) FIG. 5B depicts how the methods in this disclosure can be used to discover stabilizing commensal strains. Those strains that when present, are correlated to the longevity of the applied consortia, would become candidates for expanding the initial consortia to produce formulations with increased stability and efficacy 701.301 CLEAN COPY

Example 5: Association Study with Disease and Healthy Controls Subjects (Prophetic Example)

[0233] Comparison experiments were conducted to determine to if the present methods of the disclosure provide more insights to correlation of a microbiome that can distinguish healthy from diseased microbiome profiles over traditional current methods.

[0234] Nucleic acids were extracted from biological samples from the human microbiome. Briefly, removal of the exonuclease was conducted followed by adapter ligation at various higher concentrations. Next, size-selection using Ampure and Blue Pippen approaches were performed to enrich for the expected length amplicon species (e.g. full length 16S is about 1500 bp). After the samples were selected for the proper size, amplification reactions were conducted to prepare the libraries for sequencing. Forward and reverse primers were selected based on empirical data that indicate which sets had minimal self-complimentarity. Finally samples were sequenced using long read length sequence technology.

[0235] Using the methods above we expect to identify at set of microbes in a microbiome that would distinguish healthy from diseased subjects. The following protocol, as provide herein, can be used to assess the differences between healthy from diseased individuals. Example of diseases that can be used with the methods described include IBD, preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type 2 diabetes, C. difficile, IBS, asthma, autism, psoriasis, allergies, cardiovascular disease, cancer, depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, type 1 diabetes, halitosis, and tooth decay.

[0236] FIGS. 7A and 7B show the expected results of the comparison association study. FIG. 7A depicts the expected results from an association study with a disease and healthy controls patients' microbiomes using current technology, indicating no obvious correlation of a microbiome profile with a disease state.

[0237] In contrast, FIG. 7B depicts the expected results from an association study with disease and healthy controls patients' microbiomes using a method described herein revealing a correlation of a microbiome that can distinguish healthy controls' microbiome profiles from disease subjects' microbiome profiles (as shown FIG. 8).

[0238] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Sequence CWU 1

1

645116DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 1tgatatgtag cacgta 16216DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 2gtgatgtatc tgctcg 16316DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 3tcacagtagt cgcgag 16416DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 4tctatctctc atcgcg 16516DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 5tcgtgcgcac acactg 16616DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 6gatacacgca gatgct 16716DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 7catagtctat agagat 16816DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 8gcgactctga gtacac 16916DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 9cgacgcgcgc gactgc 161016DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 10actcgtagtg tgtcat 161116DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 11tatgcgtcag atcaga 161216DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 12ctgcatgcac gtgtac 161316DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 13acgtgagaga cgcatc 161416DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 14atcacacatc acacat 161516DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 15agctcgctct ctctga 161616DNAArtificial SequenceDescription of Artificial Sequence Synthetic barcode oligonucleotide 16cgagatatat gatgcg 161720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17agagtttgat cctggctcag 201820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18agagtttgat catggctcag 201920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 19agagtttgat cctggcttag 202020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20agaatttgat cttggttcag 202117DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21aaggaggtga tccagcc 172221DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 22acggttacct tgttacgact t 212321DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23acggctacct tgttacgact t 212421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24acggatacct tgttacgact t 212515DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 25cygaatgggg vaacc 152619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26tactdagatg tttcasttc 192718DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27cctttccctc acggtact 182819DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 28cgacatcgag gtgccaaac 192915DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 29gccaaggcat ccacc 153017DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 30cacgtctttc atcgsct 173123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 31gcgatttcyg aaygggraaa ccc 233216DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 32ccgaatgggg vaaggg 163315DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 33ccttgcccca ttcgg 153420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 34ggaactgaaa catctaagta 203516DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 35kttcgctcgc crctac 163616DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 36agtagyggcg agcgaa 163720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 37cggtactggt tcactatcgg 203822DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 38ttcgcctttc cctcacggta ct 223917DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 39agtaccgyga gggaaag 174019DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 40cattmtacaa aaggyacgc 194119DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 41cattmtrcaa aaggyacgc 194218DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 42ttcggrgaga acsagmta 184318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 43tagctggttc tcyycgaa 184416DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 44tctgggytgt tyccct 164520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 45ggatgttggc ttagaagcag 204617DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 46gttggcttrg argcagc 174715DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 47rgtgagctrt tacgc 154815DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 48wgcgtaayag ctcac 154915DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 49ggtagrrgag cgttc 155016DNAArtificial SequenceDescription of Artificial Sequence Synthetic primermodified_base(9)..(9)a, c, t, g, unknown or other 50gaggccgana rgcgta 165120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 51ggacaacagg ttaatattcc 205216DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 52aaaccgwcac aggtrg 165320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 53ggggccattt tgccgagttc 205419DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 54ccttmtcscg aasttacgg 195520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 55cttaggaccg ttatagttac 205619DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 56cgacaaggaa tttcgctac 195717DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 57gacgyaaaga ccccrtg 175817DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 58accgccccag thaaact 175918DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 59tcgctcaacg gataaaag 186015DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 60gagycgacat cgagg 156120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 61cgacgttctg aacccagctc 206217DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 62aramcgtcgt gagacag 176316DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 63agtacgagag gaccgg 166416DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 64cttagatgcy ttcagc 166514DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 65gyttagatgc yttc 146620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 66cccgcttaga tgctttcagc 206714DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 67ytgaargcat ctaa 14686DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 68ctgaag 6696DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 69gaagac 6707DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 70cctcagc 7715DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 71ccatc 5726DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 72ctggag 6736DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 73cttgag 6746DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 74cccagc 6756DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 75cgtctc 6766DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 76acctgc 6776DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 77cacgag 6786DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 78gcagtg 6796DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 79ctcttc 6805DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 80cccgc 58118DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 81tagggataac agggtaat 18825DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 82gaaga 5836DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 83gccgag 6848DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 84gcggccgc 8857DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 85gctcttc 78630DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 86tggcaaacag ctattatggg tattatgggt 308720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 87gtcgaacggt macargaaga 208820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 88grtcatggct cagattgaac 208920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 89attgaacgct ggcggcaggc 209020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 90acatgcaagt cgaacggtaa 209120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 91atcatggctc agattgracg 209220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 92attgaagagt ktgatcatgg 209320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 93catggctcag attgaamgct 209420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 94tcgaacggyw acaggaagaa 209520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 95acgctgkcgg caggcctaac 209620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 96ctmtttgctg acgagtggcg 209720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 97acggtwacwg gaagaagctt 209820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 98tgaacgctgg cggcaggcct 209920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 99ctgacgagtg gcgsacgggt 2010020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 100atrcaagtcg aacggtaaca 2010120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 101gagtttgayc atggctcaga 2010220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 102cggcaggcct aacacatgca 2010320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 103catggmycag attkaaygct 2010420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 104aacgctggcg gcaggcctaa 2010520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 105aagcttgskc tttgctgacg 2010620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 106cttgctcttt kctgacgagt 2010720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 107gctygctctt tgctgacgag 2010820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 108aaattgaagw gtttgatcat 2010920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 109gagtggcgra cgggtgagta 2011020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 110atgcaagtcg aacggtaaca 2011120DNAArtificial SequenceDescription of Artificial

Sequence Synthetic primer 111gtcgaacggt aacaggwaga 2011220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 112tggctcagat tgmacgctgg 2011320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 113gctgacgagt ggcggacggg 2011420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 114ctttgctgac gagkggcgga 2011520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 115aagaagcttg ctctttgctg 2011620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 116gaagaagctt gctctttgct 2011720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 117tttgatcatg gctmagattg 2011820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 118cgagtgscgg acgggtgagt 2011920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 119mgtggcggac gggtgrgtaa 2012020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 120agaagcttgc tctttgctgm 2012120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 121gctgacgagt ggcggwcggg 2012220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 122ctctttgctg acgagtggcg 2012320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 123ttgctgacga gtggcggacg 2012420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 124ctcagattga acrctggcgg 2012520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 125gcttgctcyt tgctgacgag 2012620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 126ttgctcttwg ctgacgagtg 2012720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 127rgcttgctct ttgctgacga 2012820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 128kggcggcagg sctaacacat 2012920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 129aggaagaagc ttgctctttg 2013020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 130tttgatcatg gctcagattg 2013120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 131acacatscaa gtcgaacggt 2013220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 132ttgctctttg ctgacgrgtg 2013320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 133aagragcttg ctctttgctg 2013420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 134ctttgctgac gagtggcgga 2013520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 135cmagtcgaac ggtaacagga 2013620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 136aagagtttga tcatggctca 2013720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 137gtaacaggaa gaagcttgct 2013820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 138saagtcgaac ggtaacmrga 2013920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 139caggaagaag cttgctcttt 2014020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 140cwgacgagtg gcggacgggk 2014120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 141aagtcgwamg gtaacaggaa 2014220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 142gastggcgga cgggtgagta 2014320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 143gytgasgagt ggcggacggg 2014420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 144gcagscctaa cacatgcaag 2014520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 145tctttgctga cgagtrgcgg 2014620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 146ctaacacatg caagtcgaac 2014720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 147gatcawggct cagattgaac 2014820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 148atcatggctc rgattgaacg 2014920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 149cgaacggtaa caggaagaag 2015020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 150tcatggctcw gattgaacgs 2015120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 151agaagcttgc tctttgctga 2015220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 152catgyaagtc gaacggtaam 2015320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 153aagartttga tcatgkctca 2015420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 154cacatgcaag tcgaacggwa 2015520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 155aacggtaaca sgaagaagct 2015620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 156gtcgaacggt aacaggaara 2015720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 157gagtttgatc atgkctcaga 2015820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 158caagtcraac ggtaacagga 2015920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 159gcctaacaca tgcaagtcga 2016020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 160aygcargtcg aacggtaaca 2016120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 161sagattgaac gctggcggca 2016220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 162gattgaacsc tggyggcmgg 2016320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 163acgagtggcg gacgggtgag 2016420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 164gaagagtttg atcatggctm 2016520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 165tcgaackgta acaggaagaa 2016620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 166ggcaggccta acacatkcaa 2016720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 167catggctcag attgaacgct 2016820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 168tggctcagat tgaacgctgg 2016920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 169gaagcttgct ctttgctgac 2017020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 170gkctcagatt gaacgctggc 2017120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 171gctggcggca ggcctaacac 2017220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 172ggmctwacac atgcragtcg 2017320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 173crstggcggc aggcctaaca 2017420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 174aggmagaagc ttgctcttwg 2017520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 175caagtcgaac ggtaacagga 2017620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 176mgcttgctct ttgctgacga 2017720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 177traagagttt gatyatggct 2017820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 178agtcgaacgg tamcagraag 2017920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 179acacatgcaa gtygaacggt 2018020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 180aagycgaacg rtaacrggaa 2018120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 181ctaagcgyac acggtggatg 2018220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 182tcggtaaggt gatatgaacc 2018320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 183ccctggsagt cagaggcgat 2018420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 184atgaaccgtt ataaccggcg 2018520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 185cagtcagagg cgatgaagga 2018620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 186cygttataac cggsgatttc 2018720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 187agcgacwaag cgtacacggt 2018820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 188ycagaggcga tgaaggacgt 2018920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 189taatcygcga taagcgtcsg 2019020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 190tgakatgaac cgttataacc 2019120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 191cacggtkkat gccctggcag 2019220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 192gcgtacacgg tggatgccct 2019320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 193taagcgtcgg waaggtgata 2019420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 194ywcacggtgg atgccctggc 2019520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 195ggcagtcaga ggcgatgaag 2019620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 196gtcggtaagg tgatatgawc 2019720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 197aggtgatatg aaccgttatr 2019820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 198wggcagtcag aggcgatraa 2019920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 199cggtggatgc cctggcagtc 2020020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 200ttataaccgg cgatttcyga 2020120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 201gcgtcggtaa ggtgatatga 2020220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 202accgttataa ccggcgattt 2020320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 203tmatctgcga taagcgtcgg 2020420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 204gcgtacacgg tggrtgccct 2020520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 205tcagaggcga tgaaggacgt 2020620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 206tamgcgtcgg taargtkata 2020720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 207atatgaaccg ttataaccgg 2020820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 208tatarccggc gatttccgaa 2020920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 209tataaccggc gatttccgaa 2021020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 210taargtgata tgaaccgkta 2021120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 211tgccctggca gtcagaggcg 2021220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 212tmaggtgata tsaaccrtta 2021320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 213gttargcgac taagcgtaca 2021420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 214tatgraccgt tataaccgrc 2021520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 215cagtcagagg cgrtgaagsa 2021620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 216cagaggcgat gaaggacgtg 2021720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 217atgccctggc agtcasaggc 2021820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 218atctgcgata agcgtcggta 2021920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 219aggacgtgct aatctgcgat 2022020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 220tctgcgataa gygtcggtaa 2022120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 221cgactaagcr tacacggtgg 2022220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 222ggcagtcaka ggsgawgaag

2022320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 223cgactaagcg twcacggtgg 2022420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 224taaggtgaka tgaaccgtka 2022520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 225ccctggcagt cagaggcgat 2022620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 226ggtaakgtga katgaaccgt 2022720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 227ttataaccgg cgatttccgr 2022820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 228gaaccgttat aaccggcgrt 2022920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 229gtgctaatct gcgataagcg 2023020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 230cacggtggat gccctggcag 2023120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 231cgactaagcg tacrcgstgg 2023220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 232acggyggatg cmctggcagt 2023320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 233atgaaggacg tgctamyctg 2023420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 234aagygtacac ggtggatgcc 2023520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 235tgaaggacgt gctaatctgc 2023620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 236rttaagcgac tmagcgtaca 2023720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 237gtgatatgaa ccgttataac 2023820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 238agcgactaar cgtacacggt 2023920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 239aaccgttata accggcgatt 2024020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 240gtacacggtg gatgccctgg 2024120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 241ggttaagcsa ctaagcgtac 2024220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 242tgcgataagc gtcggtaagg 2024320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 243aaggacgtkc taatctgcga 2024420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 244gatgccctgg magtcakagg 2024520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 245atgaaggacg tgcyaatctg 2024620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 246atgccctggc agtcagaggc 2024720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 247gtgatatsaa scgttataac 2024820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 248ccgttayaac cggcgatttc 2024920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 249gtgctaatct gcgataakcg 2025020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 250aagrwcgtgs taatctgcga 2025120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 251aatctgcgat aagcgtcggt 2025220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 252ygataagcgt cggtaaggtg 2025320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 253gaaccgttat aaccggcgat 2025420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 254gataascgkc ggtaaggwga 2025520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 255gytaatctgc gataagcgyc 2025620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 256sgtcggtaag gtgatatgaa 2025720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 257tgaaccgtta taaccggcga 2025820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 258gtgcyratct gcgataagcs 2025920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 259ctgsgakaag cgtcggtaag 2026020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 260tgcgataagc gtcggtaarg 2026120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 261tggcagtcag aggcgatgra 2026220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 262gatgaaggac gtgctaatct 2026320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 263mgmggcgatg aaggacgygc 2026420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 264ctggcagtca gaggcgatga 2026520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 265tgaaggamgt gctaatctgm 2026620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 266gaggcgatga aggacgtgct 2026720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 267ataagcgtcr gtaaggtgat 2026820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 268ggatgccctg gcagtcagag 2026920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 269ggtwaggtga tatgaaccrt 2027020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 270caccatggga gtgggttgca 2027120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 271tgactggggt gaagtcgtaa 2027220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 272cyttgtgatt catgactrgg 2027320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 273tcacaccatg ggagtgggtt 2027420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 274ggtagcttaa ccttcgggag 2027520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 275ggagggcgct taccactttg 2027620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 276tgggagtggg ttgcaaaaga 2027720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 277gagtgggttg craaagwagt 2027820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 278gackggggtg aagtcgtwac 2027920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 279cacaccatrg gaktgggktr 2028020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 280tmgtaacaag gtaaccgtag 2028120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 281tagcttaacc ttcgggaggg 2028220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 282rccactttrt gattcatgac 2028320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 283ccatgggagt gggttgcaaw 2028420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 284gagwgggttg caaaagaagt 2028520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 285cccgtcacac catggsagtg 2028620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 286gggtgaagtc gtaacaaggt 2028720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 287aagaagtagg tagcttwacc 2028820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 288camcatggga gtgrgtygca 2028920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 289aagtaggtag cttaaccttc 2029020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 290acaccatggg agtgggttgc 2029120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 291ggagtgggtt gcaaaagaag 2029220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 292sgagggcgst taccactttg 2029320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 293atgggagtsg gttgcaaaag 2029420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 294gtaacaaggt aaccgtaggg 2029520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 295gattcatgac tggggtraag 2029620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 296tgggttgcra aagaagtagg 2029720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 297acaaggtaac cgtaggggaa 2029820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 298ccatgggagt gggttgcaaa 2029920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 299gcttaacctt cgggagggcg 2030020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 300gaagtcrtaa caaggtracc 2030120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 301ttwccacttt gtgattcatg 2030220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 302ctttgtgaty catgactggg 2030320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 303catkactggg gtgaagtsgt 2030420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 304tgtgaytcat gactggggtg 2030520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 305agtcgtaaca aggtaaccgt 2030620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 306tgattcatga ctggggtkaa 2030720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 307tgtgattcat gactkgggtg 2030820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 308ccwckttgtg atwcatgact 2030920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 309gtcacaccat gggagtgggw 2031020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 310tagctkaacc ttcgggaggg 2031120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 311gcttaccact ttgtgattca 2031220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 312ttgtgattca tgactggggt 2031320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 313kttgcaaaag aagtaggtag 2031420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 314waagaagtag gtarctyaac 2031520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 315taccactkts tgattcatga 2031620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 316ccgtcacacs atgggagtgg 2031720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 317gtaacaaggw aaycgtaggg 2031820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 318rcaaggtaam cgtaggggaa 2031920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 319ggrgggcgct kascactttg 2032020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 320ytcatgactg gggtgaagtc 2032120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 321cttcrkgakg gsgcktacca 2032220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 322taccactttg trattcatga 2032320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 323gtasctwaac cttsgggagg 2032420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 324ggggtgaagw cgtaacaagg 2032520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 325aaaagragta ggtagcttaa 2032620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 326attcatgact ggggtgaagt 2032720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 327rgaagtaggt rgcttaacct 2032820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 328tagcttaacc ttcggsaggk 2032920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 329gactggggwg aagtcgtaac 2033020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 330cttaaccttm gggagggcgc 2033120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 331gactggggtg aagtcgtaac 2033220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 332gtaggtagct taaccttcgg 2033320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 333gggcgcttac cmctttgtga 2033420DNAArtificial SequenceDescription of Artificial Sequence Synthetic

primer 334aagtagrtag cttaacsttc 2033520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 335actgkggtga wgtcgtaaca 2033620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 336tgattcatga ctggggtgaa 2033720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 337wtgcaaaaga astaggtagc 2033820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 338actttgtgat tcatgactgg 2033920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 339gttgcaaaag aagtaggtag 2034020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 340gagggcgctt accactttgt 2034120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 341gggmgctyac cactttgtga 2034220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 342ttaaccttcg ggasggcgct 2034320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 343tgactggggt gaagtcktaa 2034420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 344tgrgtwgyaa magaagtagg 2034520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 345gtaggtagct traccttcgg 2034620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 346ggggtgaagt cgtaacaagg 2034720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 347gggttgcaaa agaagyaggt 2034820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 348ttgtgattca kkactggrgy 2034920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 349gcgcttacca ctttgygatt 2035020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 350cttaaysttc gggasggcgc 2035120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 351atgggagtgg sttgcaaaag 2035220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 352taacaaggta accgtagggg 2035320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 353gtcgtaacaa ggyaaccgta 2035420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 354aagaagtagg tagcttaasc 2035520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 355cacaccatgg gagtgggttg 2035620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 356cgtcacacca tgggagtggg 2035720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 357ttcatgacyg gggtgaagtc 2035820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 358agcttawccy tcgggagggs 2035920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 359ggtagcttar ccttcgggag 2036020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 360gggttgcaaa agaagtaggt 2036120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 361tcatgactgg ggtgaagtcg 2036220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 362taaccttcgg gagggcgmtt 2036320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 363wtsggagtgg gytgcaaaag 2036420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 364ccgtcacacc atgggagtgk 2036520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 365gsgccctccm gaagrttaag 2036620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 366acttcacycc agtcatgaak 2036720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 367ccgaaggtta agctacctac 2036820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 368acwaagtggt aagcgcccyc 2036920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 369caaastggwa agcgccctcc 2037020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 370cttgttacga cttcacccca 2037120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 371wttgcaaccc actcccatgg 2037220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 372ggtaagcgcc ctcccgaagg 2037320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 373actcccatgg tgtgacgggc 2037420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 374cttcacccca gtcatgaatc 2037520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 375ctacctactt cttttgcamc 2037620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 376saatcwcaaa gtggkaagcg 2037720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 377acctacytct tttgcaaccc 2037820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 378cccctacggt taycttgtta 2037920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 379gtggtaagcg ccytccckaa 2038020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 380ttctwttgca acccwctccc 2038120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 381ccgraggtta agctacctac 2038220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 382ccctcccgaa ggttaagcta 2038320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 383cctacttctt ttgcaaccya 2038420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 384cgaaggttaa gctacctact 2038520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 385rtgaatcaca aagtggtaag 2038620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 386ctcccgaagg ttaagctacc 2038720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 387tacgacttca ccccagtcat 2038820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 388tacggttacc ttgttacgac 2038920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 389ctacktcttt tgcaamccac 2039020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 390gctacctact tcttttscam 2039120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 391gtaagcgcmc tcccgaaggt 2039220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 392aagcgcmctc ccgaaggtta 2039320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 393actcccatgg tgtgacsggc 2039420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 394ttgttacgac ytcwccccag 2039520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 395aatcacaaag tggtaagcgy 2039620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 396tsacaaagtg gtaagcgccc 2039720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 397cagtcatgaa tcacaaagtg 2039820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 398tttkcaaccc actcscatgg 2039920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 399catkaatcac aaagtggtaa 2040020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 400aaggttaagc tacctacytc 2040120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 401ttcaccccag tcatgaatca 2040220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 402ggttaagcta cstacttctt 2040320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 403caacccwctc ccatggtgtg 2040420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 404acctacttct tttgcaaccc 2040520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 405acggttacct tgttacgact 2040620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 406taagcgccct cccgaaggtt 2040720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 407tcaccccagt catgaatcac 2040820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 408gtaagcgccc tcccgaaggt 2040920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 409acsacttcac cccagtcatg 2041020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 410caacccactc ccatggtgtg 2041120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 411agtcatgaat cacaaastgg 2041220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 412ttaccttgtt wcgacttcac 2041320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 413gttacgamtt caccccagtc 2041420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 414actccmatgg tgtsacgggc 2041520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 415tcaccscagt catgaatcac 2041620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 416gsaacccact cccatggtgy 2041720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 417rttacgactt caccccagtc 2041820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 418gtcatgaatc acaaagtgkt 2041920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 419tcacaaagtg gtaagcgccc 2042020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 420cttttgcaay ccactcccat 2042120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 421tcccgaargt taagctacct 2042220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 422catgaaycac aaagtggtaa 2042320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 423cccctacggt taccttgkta 2042420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 424ctacctmctt cttttgcaac 2042520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 425ttttgcracc cactcccrtg 2042620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 426tcatgaatca caaagtggtw 2042720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 427cacaaagtgk taagcgccct 2042820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 428aaggytaagc tayctacttc 2042920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 429cttttrcaac ccactcccat 2043020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 430ttttgcaasc cactcccatg 2043120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 431ctacttcttt tgsaacccac 2043220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 432ytcacmccag tcatgaatca 2043320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 433acgacttcac cccagtcatg 2043420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 434aagcgccctc ccgaaggtta 2043520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 435gcaacccact cccatggtgt 2043620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 436cctacttctt wtscaaccca 2043720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 437agtggtaagc gccmtcccga 2043820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 438cscagtcats aakcacaaag 2043920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 439crtgaatcam aaagtggtaa 2044020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 440gtcatgaatc acamagtggt 2044120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 441rgctacctac ttcttttgcm 2044220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 442ttaagctacc tacttctttt 2044320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 443cagtcrtgam tcacaaagkg 2044420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 444tcttttgcra cccactcccr 2044520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 445gtaagcgcsc tcccgaaggt

2044620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 446ggttaagsta cctacttctt 2044720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 447cccctamggt taccttsttr 2044820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 448tgttacgact tcaccccakt 2044920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 449taagcwacct acttcttttg 2045020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 450cttcttttgc aacccactcc 2045120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 451gaaggttaag ctacctactt 2045220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 452taccttgtta cgacttcacy 2045320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 453tcccctacgg ttaccttgtt 2045420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 454gttaccwtgt tacgacttca 2045520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 455cactyccatg gtgtgacggg 2045620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 456tcacaaastg gtaagcgccc 2045720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 457ccctacggtt accttgttac 2045820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 458tcccctacgg ttamcttgtt 2045920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 459ccctcccgaa ggktaagcta 2046020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 460cctcccgaag gttaagctac 2046120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 461gtaagcsccc tcccgaaggt 2046220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 462ctaagcgyac acggtggatg 2046320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 463tcggtaaggt gatatgaacc 2046420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 464ccctggsagt cagaggcgat 2046520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 465atgaaccgtt ataaccggcg 2046620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 466cagtcagagg cgatgaagga 2046720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 467cygttataac cggsgatttc 2046820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 468agcgacwaag cgtacacggt 2046920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 469ycagaggcga tgaaggacgt 2047020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 470taatcygcga taagcgtcsg 2047120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 471tgakatgaac cgttataacc 2047220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 472cacggtkkat gccctggcag 2047320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 473gcgtacacgg tggatgccct 2047420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 474taagcgtcgg waaggtgata 2047520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 475ywcacggtgg atgccctggc 2047620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 476ggcagtcaga ggcgatgaag 2047720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 477gtcggtaagg tgatatgawc 2047820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 478aggtgatatg aaccgttatr 2047920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 479wggcagtcag aggcgatraa 2048020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 480cggtggatgc cctggcagtc 2048120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 481ttataaccgg cgatttcyga 2048220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 482gcgtcggtaa ggtgatatga 2048320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 483accgttataa ccggcgattt 2048420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 484tmatctgcga taagcgtcgg 2048520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 485gcgtacacgg tggrtgccct 2048620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 486tcagaggcga tgaaggacgt 2048720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 487tamgcgtcgg taargtkata 2048820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 488atatgaaccg ttataaccgg 2048920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 489tatarccggc gatttccgaa 2049020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 490tataaccggc gatttccgaa 2049120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 491taargtgata tgaaccgkta 2049220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 492tgccctggca gtcagaggcg 2049320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 493tmaggtgata tsaaccrtta 2049420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 494gttargcgac taagcgtaca 2049520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 495tatgraccgt tataaccgrc 2049620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 496cagtcagagg cgrtgaagsa 2049720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 497cagaggcgat gaaggacgtg 2049820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 498atgccctggc agtcasaggc 2049920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 499atctgcgata agcgtcggta 2050020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 500aggacgtgct aatctgcgat 2050120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 501tctgcgataa gygtcggtaa 2050220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 502cgactaagcr tacacggtgg 2050320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 503ggcagtcaka ggsgawgaag 2050420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 504cgactaagcg twcacggtgg 2050520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 505taaggtgaka tgaaccgtka 2050620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 506ccctggcagt cagaggcgat 2050720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 507ggtaakgtga katgaaccgt 2050820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 508ttataaccgg cgatttccgr 2050920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 509gaaccgttat aaccggcgrt 2051020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 510gtgctaatct gcgataagcg 2051120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 511cacggtggat gccctggcag 2051220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 512cgactaagcg tacrcgstgg 2051320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 513acggyggatg cmctggcagt 2051420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 514atgaaggacg tgctamyctg 2051520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 515aagygtacac ggtggatgcc 2051620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 516tgaaggacgt gctaatctgc 2051720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 517rttaagcgac tmagcgtaca 2051820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 518gtgatatgaa ccgttataac 2051920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 519agcgactaar cgtacacggt 2052020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 520aaccgttata accggcgatt 2052120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 521gtacacggtg gatgccctgg 2052220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 522ggttaagcsa ctaagcgtac 2052320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 523tgcgataagc gtcggtaagg 2052420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 524aaggacgtkc taatctgcga 2052520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 525gatgccctgg magtcakagg 2052620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 526atgaaggacg tgcyaatctg 2052720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 527atgccctggc agtcagaggc 2052820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 528gtgatatsaa scgttataac 2052920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 529ccgttayaac cggcgatttc 2053020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 530gtgctaatct gcgataakcg 2053120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 531aagrwcgtgs taatctgcga 2053220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 532aatctgcgat aagcgtcggt 2053320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 533ygataagcgt cggtaaggtg 2053420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 534gaaccgttat aaccggcgat 2053520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 535gataascgkc ggtaaggwga 2053620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 536gytaatctgc gataagcgyc 2053720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 537sgtcggtaag gtgatatgaa 2053820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 538tgaaccgtta taaccggcga 2053920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 539gtgcyratct gcgataagcs 2054020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 540ctgsgakaag cgtcggtaag 2054120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 541tgcgataagc gtcggtaarg 2054220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 542tggcagtcag aggcgatgra 2054320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 543gatgaaggac gtgctaatct 2054420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 544mgmggcgatg aaggacgygc 2054520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 545ctggcagtca gaggcgatga 2054620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 546tgaaggamgt gctaatctgm 2054720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 547gaggcgatga aggacgtgct 2054820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 548ataagcgtcr gtaaggtgat 2054920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 549ggatgccctg gcagtcagag 2055020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 550ggtwaggtga tatgaaccrt 2055120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 551ttcatatcac yttaccsayg 2055220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 552acgcttatcg sagattagca 2055320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 553tagcacgtcm ttcatcgcct 2055420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 554accgaygctt atcgcagatt 2055520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 555ytcatatcac cttaccsacg 2055620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 556ccttcatcgc ctctgactgc 2055720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer

557ccaccgtgta cgcttagtcg 2055820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 558cggttataac ggttcatatc 2055920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 559ctgactgyca sggcatccac 2056020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 560gcacgtcctt catcgcctct 2056120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 561gcsggttawa acggttcata 2056220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 562acggttsatm tcaccttacc 2056320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 563ttataasggt tcatatcacc 2056420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 564cggttcatat caccttmccg 2056520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 565cttaccgacg cttatcgcag 2056620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 566gggcatccrc cgtgtacgct 2056720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 567tagcacgtcc wtcatcgcct 2056820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 568cgtgtacgct twgtcgcttw 2056920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 569racggktcat atcaccttac 2057020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 570cggttataac grttcatatc 2057120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 571ggttcatatc accttaccga 2057220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 572cctctgactg ccagggcatc 2057320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 573ttcatatcac cttacygacg 2057420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 574tctgackgcc aggrcatcca 2057520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 575ccaccgtgta cgcttagycg 2057620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 576cgacgcttat cgcagattag 2057720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 577atcgcctctg actgccaggg 2057820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 578wgcacgtcct tcatcgcctc 2057920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 579aacggttsat atcaccttac 2058020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 580catcgcctct gmckgccagg 2058120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 581ttaccgacgc ttatcgcaga 2058220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 582tcatcgcctc wgactgccag 2058320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 583accttaccga cgcttatcgc 2058420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 584gttcatatca ccttaccgam 2058520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 585attagcrcgt ccttcatcgc 2058620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 586cgtccttcat cgcctctgac 2058720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 587tcaccttacc gacgcttatc 2058820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 588ctctgactgc cagggcatcc 2058920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 589crgttataac ggttcatatc 2059020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 590tkccagggca tccaccgtgt 2059120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 591gactgccmgg gcatccamcs 2059220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 592atatcacctt ascgacgcwt 2059320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 593agggcatcca cmgtgtacgc 2059420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 594gccagggcat ccaccgtgta 2059520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 595gcagattagc acgtccttca 2059620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 596gyttatcgca gattarcacg 2059720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 597gcakattagc acgtccttca 2059820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 598aacggttcat atcaccttac 2059920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 599kacgcttatc gcwgattagc 2060020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 600ttacygacgc ttatcgcaga 2060120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 601cgcttatcgc agattagcac 2060220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 602cgtccttcat cgmctctgrc 2060320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 603twaccgacgc ttatcgcaga 2060420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 604gccggttata acggttcaya 2060520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 605gcatmcaccg tgtaygstta 2060620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 606cggmaatcgc cggttataac 2060720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 607ccgtgtacgc ttagkcgctt 2060820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 608ktaccgacgc ttatcgcaga 2060920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 609cgcttatcgc agattmgcac 2061020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 610acggttcata tcaccytacc 2061120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 611cctctgwctg ccagggcatc 2061220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 612gcttrtcgca gattagcacg 2061320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 613ggcatccacc gtgtacgctt 2061420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 614ggcatccacc stgtacgctt 2061520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 615tatsacctta ccgacgctta 2061620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 616tmgcmgatta gcacgtsmtt 2061720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 617tcgcagatta gcaygtcctt 2061820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 618tatcayctta ccgaygctta 2061920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 619acgtccttca tcgcctctga 2062020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 620gggcatccac cgtgtacgct 2062120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 621acggttcata tcaccttmcc 2062220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 622agggcatccr ccgtgtacgc 2062320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 623catakmrcct trccgacsct 2062420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 624gacgcttatc gcakattagc 2062520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 625tccttcatcg cctctgactg 2062620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 626gttcatatcm ccttrccgac 2062720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 627tcggaaatcg ccggktataa 2062820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 628acgcttatcg cagattagca 2062920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 629gttcatatca ccttaccgac 2063020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 630gycagggcat mmacmstgta 2063120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 631tcacyttacc gacscttakc 2063220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 632tagcacgtcs ttcatcgcct 2063320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 633tcgcctctga ckgccagggc 2063420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 634gggcatccmc cgtgtacgct 2063520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 635ggttcrtatc acmttaccga 2063620DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 636atcgcagatt agcacgtcct 2063720DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 637tcgccggtta taackgttca 2063820DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 638agcacgtcct tcatcgcctc 2063920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 639gcttatcgca gattagcrcg 2064020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 640tatcaccttw ccgacgctta 2064120DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 641cggttcatat cascttaccg 2064220DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 642atcgcctctg rmtgcyaggg 2064320DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 643tcyaccgtgt acgcttagwc 2064420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 644tcgcagatta gcacgtcctt 2064520DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 645tscaccgtgt acgcttagtc 20

Claims

1-38. (canceled)

39. A method of profiling a microbiome by assaying a biological sample of a subject, the method comprising: (a) obtaining said biological sample from said subject, wherein said biological sample comprises a plurality of microbes, wherein said subject suffers or is at risk from suffering from a condition; (b) subjecting nucleic acid molecules extracted from said plurality of microbes to sequencing to obtain nucleic acid sequence reads that comprise ribosomal RNA operon sequences from said plurality of microbes, wherein at least one of said nucleic acid sequence reads provides a contiguous sequence spanning at least a 16S region of said ribosomal RNA operon, a 23 S region of said ribosomal RNA operon, or an intergenic region between said 16S region and said 23S region of said ribosomal RNA operon; (c) aligning said nucleic acid sequence reads of said ribosomal RNA operon sequences to one or more reference sequences to identify said plurality of microbes at a strain level; (d) using a database to identify metabolic pathways that correspond to said plurality of microbes identified at said strain level in (c); and (e) based on (d), administering a microbial-based composition to said subject, thereby treating the condition.

40. The method of claim 39, wherein at least one of said nucleic acid sequence reads comprises said contiguous sequence spanning at least said 16S region.

41. The method of claim 39, wherein at least one of said nucleic acid sequence reads comprises said contiguous sequence spanning at least said 23 S region.

42. The method of claim 39, wherein at least one of said nucleic acid sequence reads comprises said contiguous sequence spanning said intergenic region between said 16S region and said 23 S region of said ribosomal RNA operon.

43. The method of claim 39, wherein said plurality of microbes in said microbiome profile comprises 800 or fewer microbes.

44. The method of claim 39, further comprising obtaining said nucleic acid molecules from said plurality of microbes in biological samples taken at least at two different points of time.

45. The method of claim 39, wherein a presence or absence of said plurality of microbes and said metabolic pathways is indicative of said subject having said type 2 diabetes.

46. The method of claim 39, further comprising detecting or measuring an amount of said nucleic acid sequence reads of said plurality of microbes.

47. The method of claim 46, wherein said detecting or measuring said amount of said nucleic acid sequence reads of said plurality of microbes comprises detecting levels of markers from said plurality of microbes.

48. The method of claim 39, wherein at least one of said nucleic acid sequence reads provides a contiguous sequence spanning said 16S region and said 23S region of said ribosomal RNA operon.

49. The method of claim 39, wherein each of said nucleic acid sequence reads is longer than 2000 bases.

50. The method of claim 39, wherein said sequencing of (b) is performed using a long read sequencing platform.

51. The method of claim 39, further comprising extracting said nucleic acid molecules from said plurality of microbes in said biological sample.

52. The method of claim 39, wherein the condition is selected from the group consisting of inflammatory bowel disease, preterm labor, obesity, diabetic foot ulcers, bacteremia, acne, infantile colic, type II diabetes, C. difficile, irritable bowel syndrome, asthma, autism, psoriasis, allergies, cardiovascular disease, cancer depression, cystic fibrosis, multiple sclerosis, urinary tract infection, radiation enteropathy, drug metabolism, chronic fatigue, type I diabetes, halitosis, and tooth decay.

Images