Microbiome identification and bacteriophage preparation

Abstract

Provided herein is a method for treating a health condition in a subject. The method may include detecting a microorganism in a sample from the subject, and following identification of the microorganism, generating a bacteriophage mixture comprising at least one bacteriophage capable of lysing the microorganism. One or more of the processes described herein may include shotgun metagenomic sequencing.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 163,375, filed March 19, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] A biological sample obtained from a subject may contain a variety of different microorganisms, which together may form the subject's microbiome or the subject's body (e.g., skin, gut). The microbiome may be useful in understanding one or more health parameters of the subject or in identifying one or more pathologies.

[0003] Skin microbiome may include the microbial community living in and on the skin of a subject.Methods for measuring and analyzing skin microbiome may include biological analysis, such as DNA sequencing.However, current methods for measuring skin microbiome may introduce bias during sampling, measurement, and analysis, which may lead to inaccurate diagnosis, treatment, and monitoring. Summary of the Invention

[0004] Recognized herein is a need for improved methods and systems for measuring the microbiome in a subject's body (e.g., skin). The present disclosure provides systems and methods for detecting a population of microorganisms in a biological sample obtained from a subject, as well as methods for treating one or more conditions (e.g., skin conditions) in a subject.

[0005] In one aspect, provided herein is a method of treating a skin condition in a subject, the method comprising: (a) detecting a population of microorganisms in a skin sample obtained from the subject, the detecting comprising performing shotgun metagenomic sequencing of nucleic acid molecules extracted from the population of microorganisms, thereby generating a set of sequence reads, and processing the set of sequence reads; and (b) administering to the subject a bacteriophage or fungal-virus formulation comprising at least one virus capable of lysing microorganisms of the microbial population.

[0006] In some embodiments, the set of sequence reads consists of 0.1M to 100M sequence reads. In some embodiments, the population of microorganisms comprises bacteria or fungi. In some embodiments, the method further comprises detecting one or more mites in a skin sample obtained from the subject. In some embodiments, the population of microorganisms comprises one or more microorganisms associated with acne. In some embodiments, the population of microorganisms comprises one or more microorganisms associated with inflammation, redness, eczema, rosacea, enlarged hair follicle pore size, rough skin texture, increased transepidermal water loss, skin discoloration, or unequal elasticity of the stratum corneum and underlying dermis. In some embodiments, the population of microorganisms comprises one or more microorganisms associated with aging skin. In some embodiments, the population of microorganisms comprises Propionibacterium acnes bacteria. In some embodiments, the population of microorganisms comprises Staphylococcus aureus bacteria. In some embodiments, the population of microorganisms comprises Corynebacterium bacteria. In some embodiments, the population of microorganisms comprises Malassezia yeast. In some embodiments, the detecting further comprises (1) isolating DNA from the skin sample, and (2) removing human DNA from the isolated DNA. In some embodiments, the method further comprises adding an internal DNA standard to the isolated DNA. In some embodiments, the internal DNA standard comprises synthetic DNA. In some embodiments, the processing comprises removing one or more background sequence reads from the set of sequence reads, the background sequence reads being identified by shotgun genomic sequencing of DNA isolated from the negative control sample. In some embodiments, the processing comprises normalizing the counts of the set of sequence reads to generate a set of normalized sequence reads. In some embodiments, the processing comprises clustering the set of normalized sequence reads. In some embodiments, the processing comprises comparing the set of normalized sequence reads to a database. In some embodiments, the database comprises sequences of a plurality of microorganisms isolated from other skin samples from other subjects. In some embodiments, the database comprises sequences of mobile genetic elements.In some embodiments, the mobile genetic element comprises one or more members of the group consisting of a plasmid, a prophage and a transposon. In some embodiments, the database comprises genomic sequences of microorganisms isolated from other skin samples from other subjects. In some embodiments, the mobile genetic element comprises one or more members of the group consisting of a plasmid, a prophage and a transposon. In some embodiments, the bacteriophage or fungal virus preparation does not comprise a lysogenic bacteriophage. In some embodiments, the bacteriophage or fungal virus preparation comprises a first bacteriophage and a second bacteriophage. In some embodiments, the first bacteriophage and the second bacteriophage are capable of lysing microorganisms of the population of microorganisms. In some embodiments, the first bacteriophage and the second bacteriophage have different host ranges. In some embodiments, the different host ranges comprise at least two Propionibacterium acnes strains. In some embodiments, the different host ranges comprise at least one Propionibacterium acnes strain and at least one C. namnatense strain. In some embodiments, the different host ranges do not include C. granulosum strains. In some embodiments, the first bacteriophage and the second bacteriophage individually or collectively prevent resistance or regrowth of the microorganism in vitro. In some embodiments, the first bacteriophage and the second bacteriophage are lytic bacteriophages. In some embodiments, the bacteriophage or fungal virus preparation is part of a cosmetic formulation. In some embodiments, the cosmetic formulation comprises one or more liposomes comprising the first bacteriophage and the second bacteriophage. In some embodiments, the cosmetic formulation further comprises an anti-aging component.

[0007] In another aspect, provided herein is a method of determining a microbiome type, comprising: (a) combining DNA isolated from a skin sample of a subject with an internal DNA standard to form a DNA mixture; (b) performing shotgun metagenomic sequencing of the DNA mixture to generate a set of sequence reads; and (c) processing the set of sequence reads to determine the microbiome type.

[0008] In some embodiments, the subject has acne. In some embodiments, the subject has one or more skin conditions selected from the group consisting of redness, eczema, rosacea, and aging skin. In some embodiments, the DNA isolated from the skin sample contains less than 10% human DNA. In some embodiments, the internal DNA standard is a synthetic internal DNA standard. In some embodiments, the processing comprises transforming the set of sequence reads to generate transformed sequence reads. In some embodiments, the method further comprises clustering the transformed sequence reads. In some embodiments, the processing comprises normalizing a count of a subset of the set of sequence reads to another count of the set of sequence reads from the internal DNA standard. In some embodiments, the processing comprises comparing the plurality of sequence reads to a set of sequence reads of a negative control sample, the negative control sample being open to air for a short period of time along with the skin sample and processed in parallel with the skin sample. In some embodiments, the processing comprises detecting one or more subtypes of Propionibacterium acnes. In some embodiments, the processing comprises detecting one or more subtypes of Staphylococcus aureus. In some embodiments, the processing comprises detecting one or more subtypes of Corynebacterium.

[0009] In another aspect, disclosed herein is a method that includes: (a) contacting (i) a sample from an environmental source with (ii) one or more cultured microorganisms from a skin sample; and (b) isolating a lytic bacteriophage capable of lysing the one or more cultured microorganisms; and (c) sequencing nucleic acid isolated from the lytic bacteriophage.

[0010] In some embodiments, the environmental source is sewage or a soil suspension. In some embodiments, the method further comprises identifying the one or more cultured microorganisms by a method comprising DNA sequencing. In some embodiments, the one or more cultured microorganisms comprise Propionibacterium acnes strains. In some embodiments, the one or more cultured microorganisms comprise Staphylococcus aureus strains. In some embodiments, the one or more cultured microorganisms comprise Corynebacterium strains. In some embodiments, the skin sample comprises a skin swab from skin affected by acne, inflammation, redness, eczema, rosacea, or aging. In some embodiments, the method further comprises determining the host range of the lytic bacteriophage. In some embodiments, the host range does not include beneficial bacteria associated with a low risk of a skin condition. In some embodiments, the method further comprises constructing a bacteriophage library comprising the lytic bacteriophage. In some embodiments, the bacteriophage library comprises a plurality of lytic bacteriophages capable of lysing one or more cultured microorganisms.

[0011] In another aspect, disclosed herein is a method of selecting a bacteriophage for inclusion in a bacteriophage formulation, the method comprising: (a) preparing a mixture of a first bacteriophage and a second bacteriophage; (b) contacting the mixture with a cultured microorganism; and (c) if the cultured microorganism fails to regrow after contacting, selecting the first bacteriophage and the second bacteriophage for inclusion in the bacteriophage formulation, wherein the cultured microorganism comprises a Cutibacterium bacterium, a Staphylococcus bacterium, or a Corynebacterium bacterium.

[0012] In some embodiments, the first bacteriophage and the second bacteriophage have different host ranges. In some embodiments, the first bacteriophage and the second bacteriophage are non-lysogenic bacteriophages. In some embodiments, the method further comprises (d) preparing a cosmetic formulation comprising the first bacteriophage and the second bacteriophage. In some embodiments, the cosmetic formulation comprises a liposome. In some embodiments, the first bacteriophage and the second bacteriophage are capable of lysing cultured microorganisms after storage in the cosmetic formulation for at least two months.

[0013] In another aspect, the methods provided herein include: (a) identifying a host bacterium of interest using shotgun metagenomic sequencing; (b) upon identification of the host bacterium, generating a bacteriophage mixture comprising a first bacteriophage, a second bacteriophage, and a stabilizing buffer, where both the first bacteriophage and the second bacteriophage are capable of infecting the host bacterium.

[0014] In some embodiments, the host bacterium is a Propionibacterium acnes bacterium. In some embodiments, the first bacteriophage and the second bacterium do not infect a useful strain of Propionibacterium acnes. In some embodiments, the host bacterium is a Staphylococcus aureus bacterium. In some embodiments, the first bacteriophage and the second bacteriophage do not infect Staphylococcus epidermidis. In some embodiments, the host bacterium is a Corynebacterium genus bacterium. In some embodiments, the first bacteriophage and the second bacterium do not infect a useful strain of Corynebacterium. In some embodiments, the first bacteriophage and the second bacteriophage are encapsulated in one or more liposomes. In some embodiments, the bacteriophage mixture includes a retinoid (vitamin A derivative), niacinamide (vitamin B3), ascorbic acid (vitamin C), acetate or tocopherol (vitamin E derivative), a skin active peptide, a plant growth factor such as kinetin, or ubiquinone (coenzyme Q10).In some embodiments, the first bacteriophage or the second bacteriophage specifically infects one or more members of the group consisting of Propionibacterium acnes bacteria, Staphylococcus aureus bacteria, and Corynebacterium spp. bacteria.

[0015] Another aspect of the present disclosure provides a non-transitory computer-readable medium that includes machine-executable code that, upon execution by one or more computer processors, performs any of the methods described above or otherwise herein.

[0016] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto, the computer memory including machine executable code that, when executed by the one or more computer processors, performs any of the methods described above or elsewhere herein.

[0017] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which merely illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0018] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material.

[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Brief description of the drawings]

[0020] [Figure 1] 1 shows exemplary data from a bacteriophage host range assay. [Diagram 2] 1 shows exemplary data of bacterial growth in response to contact with one or more bacteriophages. [Diagram 3] 1 illustrates a computer system programmed or otherwise configured to perform the methods provided herein. [Figure 4] 1 shows another set of exemplary data from a bacteriophage host range assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be made by those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present invention described herein may be used.

[0022] In some embodiments, systems, compositions and methods are provided herein for determining a type of microbiome from a skin sample from a subject. Determining a type of microbiome may include detecting a population of microorganisms in a skin sample obtained from a subject. Such detection may be useful for diagnosing or treating a skin condition (e.g., acne, inflammation, aging) of a subject and / or determining how a population of microorganisms or a subpopulation therein affects or acts on a skin condition. In some examples, a treatment may be administered to a subject based on the detected microbiome type. Such a treatment method may include administering to a subject a formulation that can be used to selectively lyse or inhibit the growth of a particular microorganism (e.g., bacteria) in a population of microorganisms. In some embodiments, the formulation may include at least one bacteriophage that can lyse a microorganism in a population of microorganisms. Such methods and compositions may be useful for personalized treatment of one or more skin conditions.

[0023] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to every number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0024] Whenever the terms "only", "less than", or "less than or equal to" precede the first number in a series of two or more numbers, the terms "only", "less than", or "less than or equal to" apply to each number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0025] The term "sample" as used herein generally refers to a biological sample of a subject. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a swab (e.g., from the mouth, skin, or other externally facing body). The sample may be a skin sample, such as obtained from a swab, microporous strip, skin aspirate, etc. The sample may be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. The sample may be a buccal swab. The sample may be a plasma or serum sample. The sample may be an acellular sample. The sample may be or may include a viral sample. The acellular sample may include extracellular polynucleotides. The extracellular polynucleotides may be isolated from a bodily sample, which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal secretions, sputum, stool, and tears. The biological sample may include any number of macromolecules, such as cellular macromolecules. The sample may be a cell sample. The sample may include one or more cells or viruses. The sample may include one or more microorganisms. The biological sample may be a nucleic acid sample or a protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from other samples.

[0026] The term "subject" as used herein generally refers to an animal, such as a mammal (e.g., a human) or an avian (e.g., a bird), or other organism, such as a plant. For example, a subject may be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a monkey, or a human. Animals may include, but are not limited to, livestock, sports animals, and pets. A subject may be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., cancer), or a predisposition to a disease, infection, health condition, and / or an individual in need of treatment or suspected of needing treatment. A subject may be a patient.

[0027] The term "sequencing" as used herein generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. A polynucleotide may be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA, complementary DNA, etc.). Sequencing can be performed by a variety of currently available systems, including, but not limited to, sequencing systems by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®). Alternatively or additionally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such systems can provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a human) generated by the system from a sample provided by the subject. In some examples, such systems provide sequencing reads (also "reads" herein). A read may include a series of nucleic acid bases that correspond to the sequence of a sequenced nucleic acid molecule. In some circumstances, the systems and methods provided herein may be used in conjunction with proteomic information.

[0028] Microbiome type identification and bacteriophage preparation In one aspect, provided herein is a method for determining a microbiome type or measuring a microbiome of a subject's body (e.g., skin, gut). The method of the present disclosure may include detecting a population of microorganisms in a sample (e.g., skin sample) obtained from a subject and determining a microbiome type. In some cases, detecting includes performing metagenomic sequencing (e.g., shotgun metagenomic sequencing) of nucleic acid molecules extracted from the population of microorganisms. Such a detection method may include generating a set or a plurality of sequence reads and processing the set or a plurality of sequence reads. In some aspects of the present disclosure, the method may further include administering a bacteriophage preparation to the subject, the bacteriophage preparation comprising at least one bacteriophage capable of lysing microorganisms of the population of microorganisms. The methods provided herein may be useful for identifying, diagnosing, or treating a skin condition (e.g., acne, inflammation, redness, eczema, rosacea, aging, etc.) in a subject.

[0029] Skin Microbiome: In some embodiments, the present disclosure provides a method for analyzing the skin microbiome type on a skin sample and determining one or more microorganisms. The skin microbiome may include a population of microorganisms living in and on the skin. The population of microorganisms may include the same type of microorganism, or the population of microorganisms may include different types of microorganisms. For example, the population of microorganisms may include one or more bacteria, yeast, fungi, protozoa, viruses, or combinations thereof. In some examples, the population of microorganisms may include one or more mites.

[0030] Skin Sample: A skin sample can be obtained from a subject using one or more approaches. In some examples, the skin sample is obtained using a swab. The swab can include any useful shape or material. For example, the swab can be rod-shaped and include a first end that includes a specimen collection area. The specimen collection area can be any useful shape, such as circular, triangular, pyramidal, rectangular, diamond, pentagonal, hexagonal, heptagonal, octagonal, etc. The swab can include any useful material, such as synthetic materials (e.g., polymers such as nylon, rayon, Dacron, etc.) or naturally occurring materials (e.g., cotton, bamboo, etc.). In some examples, the swab is a synthetic flocked swab. In some examples, the skin sample is obtained using a tape strip. For example, the tape strip can be a pore strip that can be used to extract contents from a pore or pilosebaceous unit. In some examples, the skin sample is obtained using a skin biopsy.

[0031] Skin sample processing: The skin sample may be processed prior to detecting one or more microorganisms. Such processing may include, for example, extracting or isolating one or more nucleic acid molecules (e.g., DNA, RNA) from the sample, and optionally removing contaminating specimens from the sample. In some examples, contaminating nucleic acid molecules (e.g., DNA, RNA) may be removed from the sample. For example, DNA from the subject or host (e.g., human) may be removed from the skin sample or separated from the DNA of other organisms in the skin sample. Alternatively or additionally, microbial DNA from the skin sample may be concentrated or purified. Such microbial DNA concentration or host DNA depletion may include techniques for lysis of human cells and / or degradation of mammalian or human DNA, such as osmotic lysis, propidium monoazide treatment, or other approaches. Thus, the processed sample may contain minimal human DNA, for example, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1% or less human DNA.

[0032] In some examples, an internal standard can be added to the isolated or extracted nucleic acid molecule. The inclusion of an internal standard (e.g., a DNA standard) can be performed by spiking a known DNA sequence into the sample (or processed sample, e.g., the isolated or extracted DNA). In some examples, the addition of an internal standard can be useful in adjusting the relative sequence read counts obtained from sequencing, or to normalize the sequence read counts, thereby improving the quantitative accuracy of the sequencing. The internal standard can be a synthetic internal DNA standard, or the internal standard can include a naturally occurring DNA molecule. The internal DNA standard can include metagenomic sequins, such as those described in Hardwick, et al. 2018. "Synthetic Microbe Communities Provide Internal Reference Standards for Metagenome Sequencing and Analysis." Nature Communications 9(1):3096, which is incorporated herein by reference in its entirety.

[0033] The inclusion of a control sample can also be used to improve the accuracy of determining microbiome subtypes. In one example, a negative control sample, such as a swab exposed to the environment or air, can be included for measuring or analyzing the microbiome. In such an example, a skin sample can be collected with a first set of swabs, and a negative control set of swabs exposed to air but not to the subject's skin can be included for further processing as described herein and analysis, such as via sequencing, to generate a set of negative control sequence reads. The negative control sequence reads, or a portion thereof, can be subtracted or removed (e.g., computationally) from the sequence reads of the skin sample. Such techniques can be useful for removing background nucleic acid contamination (e.g., removing environmental or airborne bacterial DNA).

[0034] Additional processes can be performed to prevent contamination or remove contaminants from the skin sample. For example, sterile handling techniques can be performed, such as processing the sample in a sterile laminar flow hood, sterilizing tools or reagents, treating tools or agents with enzymes (e.g., RNAse or DNAse) to remove contaminating nucleic acid molecules, and the like. As will be appreciated, any number of processing, decontamination, and / or enrichment techniques can be used to optimize the accuracy of the sample readout (e.g., by sequencing). One or more of the processing, decontamination, and / or enrichment techniques can improve the sample readout by removing or reducing bias during sample analysis, such as by sequencing, clustering sequence reads into skin microbiome types, and identifying one or more microorganisms. Thus, such sample processing can be used to improve the accuracy of the identification of the type of skin microbiome.

[0035] Measuring the skin microbiome: Determining the type of microbiome or identifying one or more microorganisms on a skin sample can be performed using DNA sequencing (e.g., high-throughput DNA sequencing) methods. Sequencing can include 16S rRNA sequencing or shotgun metagenomic sequencing. In some examples, shotgun metagenomic sequencing can be performed on the skin sample, which can allow for accurate identification of the type of skin microbiome. Shotgun metagenomic sequencing can be useful to comprehensively sample all genes present in a complex sample (e.g., containing nucleic acid molecules from multiple microbial types), thereby allowing detection or identification of one or more (or all) microorganisms present in the sample. In some examples, the use of shotgun metagenomic sequencing can be used to avoid the use of 16S rRNA sequencing, which may have limited taxonomic resolution or introduce bias into the skin microbiome analysis, for example, by excluding microorganisms or viruses that lack 16S rRNA genes or excluding microorganisms (e.g., bacterial taxa) from primer design. In other examples, the use of shotgun metagenomic sequencing can complement the use of 16S rRNA sequencing. Shotgun metagenomic sequencing can be useful for detecting non-bacterial microorganisms, such as yeasts (e.g., Malassezia yeast), mites (e.g., Demodex), etc. Advantageously, detecting non-bacterial microorganisms can increase the accuracy of profiling skin samples and correlate different microbial types with specific skin conditions (e.g., folliculitis, eczema, redness, acne, rosacea, aging skin, etc.) or skin appearance.

[0036] Shotgun metagenomic sequencing: In some examples, shotgun metagenomic sequencing can be used to generate sequence reads from one or more organisms of a sample. Shotgun metagenomic sequencing can be useful to resolve strain-level variants that may not be resolvable from 16S rRNA sequencing. For example, shotgun metagenomic sequencing can be useful to determine sequences of microbial accessory genomes that may be undetectable from 16S rRNA sequencing. Such accessory genomes may include genetic elements that are strain-specific or not essential for microbial survival, in non-limiting examples. In some examples, shotgun metagenomic sequencing can be used to resolve or detect mobile genetic elements, such as plasmids, prophages, or transposons. Mobile genetic elements may include various accessory genes that may confer traits such as antibiotic resistance, virulence, or metabolic pathways. Determining strain-level mutations of a sample can help determine how one or more microbes (e.g., bacteria such as Propionibacterium acnes or Staphylococcus aureus, yeast, protists, etc.) or microbial genetic factors contribute to or affect skin conditions (e.g., acne, aging, inflammation). Alternatively or additionally, determining strain-level mutations in a sample may allow for the determination of microbial susceptibility (e.g., small molecule therapeutics, bacteriophages, etc.) and virulence factors. Thus, information obtained from shotgun metagenomic sequencing can be used to design formulations (e.g., bacteriophage formulations) known to target specific microorganisms (such as those found on skin samples) in polymicrobial samples.

[0037] Shotgun metagenomic sequencing can also be useful to generate sufficient sequencing depth to detect rarer microbial or viral variants or strains. For example, shotgun metagenomic sequencing can capture strain-level variation in bacteriophage receptor genes, which may indicate bacteriophage susceptibility and virulence factors in skin bacteria. Thus, some useful sequencing depth can be obtained from shotgun metagenomic sequencing. For example, sequence reads generated from a skin sample can include at least 0.1 million (M), at least 1 M, at least 5 M, at least 10 M, at least 20 M, at least 30 M, at least 40 M, at least 50 M, at least 60 M, at least 70 M, at least 80 M, at least 90 M, at least 100 M, at least 200 M or more sequence reads. In some cases, sequence reads generated from a skin sample may include up to 200M, up to 100M, up to 90M, up to 80M, up to 70M, up to 60M, up to 50M, up to 40M, up to 30M, up to 30M, up to 10M, up to 5M, up to 1M, up to 0.1M or less sequence reads. A range of sequence reads, for example, from about 1M to about 100M sequence reads, may be generated from a skin sample.

[0038] Analysis of metagenomic data: After shotgun metagenomic sequencing, the sequence reads can be further processed or analyzed (e.g., computationally). Such processing or analysis can include, in non-limiting examples, filtering, assembly, binning, gene prediction, etc., and can be performed using one or more processors. In one example, the sequence reads can be pre-filtered, for example, by removing redundant sequences, low quality sequences, contaminating sequences, etc. In some cases, the sequence reads can be assembled to determine the origin (e.g., microorganism, subject, etc.) from which the sequence reads originate. Such assembly can be performed using one or more assembly programs, for example, Phrap, Celera Assembler, Velvet Assembler, etc. Alternatively or additionally, assembly can be performed by generating a de Brujin graph and assembling the reads. One or more sequence reads generated from sequencing can be compared to a reference genome, for example, for assembly, binning, gene prediction, etc. The assembled sequences or contigs can be further annotated for coding regions. Such annotation may include identification of genes based on sequence homology with known gene sequences (e.g., homology with publicly available sequence databases, e.g., BLAST, RefSeq). Any useful program for gene prediction may be implemented, such as MEGAN, GeneMark, GLIMME, MetaProdigal, FragGeneScan, MetaGeneMark, SNAP, EuGene, etc. In some examples, custom algorithms may be generated for assembly, annotation, or any other processing operation. Alternatively, no assembly may be performed and the sequences may be further processed, e.g., for taxonomic profiling, functional analysis, etc.

[0039] In some examples, assembled sequences or contigs (or annotated sequences) are binned to associate sequences with organisms. Such binning can be performed using a homology-based approach (e.g., searching for phylogenetic markers using BLAST, or other programs such as Meta Genome Analyzer (MEGAN), PhymmBL, MetaPhiAn, AMPHORA, mOTUS, MetPhyler, SLIMM, etc.). Other binning algorithms include, but are not limited to, DAS Tool MetaWRAP, CheckM, AMBER, SolidBin, CARMA, Sort-iTEMS, TACOA, PhyloPythiaS, Phymm, SCIMM, Metawatt, LikelyBin, MaxBin, COCACOLA, MetaBAT2, CONCOCT, BMC3c, MetaBMF, MBCC, Canopy.

[0040] In some examples, sequence reads or derivatives thereof (e.g., assembled reads, assembled contigs, binned reads, etc.) can be transformed. For example, proportionality can be used (e.g., applied to sequence reads) to determine the proportion of reads that are assigned to microbial taxa. Transformation can be performed in any useful processing operation, such as before, during, or after clustering or binning of sequence reads, before, during, or after assembly, etc.

[0041] Sequence reads can be normalized, for example, to determine the relative number of each sequence within a sample or to compare sequence counts across samples. Such normalization of counts can be performed, for example, by including appropriate controls during sample processing, as described elsewhere herein, for example, by including (e.g., by spiking in) a synthetic internal DNA standard in each sample or DNA library prior to sequencing. Following sequencing, the counts of each sequence read from a sample (e.g., from one or more microorganisms) can be normalized to the counts of the DNA standard to obtain the relative number of each sequence read. Thus, the relative read counts from a first sample (e.g., a first skin sample from a subject) can be compared to the relative read counts in another sample (e.g., a second skin sample from the subject or a different subject), allowing for quantitative comparison of sequence reads across samples. Beneficially, normalizing each sequence read count can improve the accuracy of quantification of sequence reads present within a sample or across multiple samples. Normalization of sequence reads can be performed at any convenient or advantageous time in sequence data analysis, e.g., before clustering, before binning, before assembling.

[0042] The sequence reads or their derivatives (e.g., normalized sequence reads) can be clustered using any useful clustering method. For example, the clustering can include random forest clustering. Clustering can be performed based on common features, such as unitigs from a colored compact de Brujin graph or hashed kmers.

[0043] Taxonomic profiling of sequence reads or derivatives thereof can be performed to, for example, identify microbial taxa (e.g., species of microorganisms in a sample). Taxonomic profiling can include or be part of annotation, or taxonomic profiling can be a separate process. Taxonomic profiling can include identification of genes based on sequence homology with known gene sequences (e.g., homology with publicly available sequence databases, e.g., BLAST, RefSeq). Any useful program can be implemented for taxonomy, e.g., MEGAN, MGS-Fast, MG-RAST, MAGpy, DIAMOND, etc. In some examples, programs used for taxonomic profiling can include open source or publicly available programs, e.g., Sourmash. Alternatively or additionally, custom sequence databases, e.g., genomic databases generated from sequencing multiple skin samples, such as those obtained using the methods described herein, can be generated and used as reference sequences for taxonomic profiling.

[0044] Microbiome Library: Skin samples from multiple subjects may be collected and subjected to sequencing (e.g., shotgun metagenomic sequencing), and the sequencing reads may be stored in a microbiome library. The microbiome library may contain sequences of multiple microorganisms obtained from skin samples, and may include DNA sequences of bacteria, yeast, mites, viruses, etc. The sequences may include genomic DNA or accessory genomic sequences, for example, from plasmids, transposons, prophages, etc.

[0045] In some examples, the sample or microorganisms from the sample may also be collected and stored. For example, one or more microorganisms may be removed, isolated, or extracted from the sample, a portion of the removed, isolated, or extracted microorganisms may be subjected to sequencing as described herein, and a portion may be cultured (e.g., collected and grown on a plate). The cultured microorganisms may be used for further screening, for example, to identify one or more agents that can lyse or inhibit the growth of at least one of the cultured microorganisms. Since different samples may contain different microbiomes and microbial species, each cultured sample may be kept separate from the cultured microbiomes of other samples. Thus, screening for agents that can lyse or inhibit the growth of one or more microorganisms in each of these samples may allow for personalized treatment of skin conditions caused by specific microbiome types. Alternatively or additionally, the microbiomes of the entire samples may be mixed together and screened to identify one or more agents that can lyse or inhibit the growth of the pooled sample.

[0046] Bacteriophage assay: A sample can be screened to determine one or more agents (e.g., small molecules, bacteriophages, or biological molecules such as proteins, peptides, lipids, carbohydrates, metabolites, or combinations thereof) that can inhibit the growth or otherwise kill (e.g., by lysis) one or more microorganisms in the sample. In some examples, the one or more agents include one or more bacteriophages. For example, the one or more bacteriophages can be naturally occurring, recombinant, or synthetic bacteriophages that can lyse certain bacteria present on the skin sample. In such examples, screening the sample can include obtaining one or more microorganisms from the sample, optionally culturing the one or more microorganisms, and exposing the one or more microorganisms to multiple bacteriophages to generate a microorganism-bacteriophage mixture. The growth, proliferation, or death of the microorganisms can be monitored over a period of time, and the microorganisms can be collected, stored, and / or sequenced. Sequencing (e.g., shotgun metagenomic sequencing) can provide information about bacteriophages present in or on microorganisms that can kill or inhibit the growth of one or more microorganisms. Thus, such screening, for example, a combination of culturing a microorganism with a bacteriophage and sequencing the mixture, can be useful to determine combinations of bacteriophages to eliminate specific microbial (e.g., bacterial) strains.

[0047] In some examples, the screening includes sequencing one or more microorganisms isolated from the sample. For example, one or more microorganisms isolated from the sample can be (i) cultured to generate a microbial library and (ii) sequenced (e.g., by shotgun metagenomic sequencing) to generate a library of sequence reads that include sequences from the microorganisms present in the sample. Additionally, the cultured microorganisms can be further exposed to one or more bacteriophages, and then isolated and sequenced, as described above. The sequences corresponding to the one or more bacteriophages can be stored in a bacteriophage library. Alternatively or additionally, one or more bacteriophages can be stored and further screened, for example, using a bacteriophage host range assay to determine bacterial strains that may be susceptible to the one or more bacteriophages.

[0048] Bacteriophage source: One or more bacteriophages may be synthetic or naturally occurring. One or more bacteriophages may be isolated or extracted from environmental sources, including, but not limited to, sewage, soil suspensions, marine sediments, and subsurface land. In such cases, further processing of the environmental source may be performed to purify or concentrate the bacteriophage.

[0049] Bacteriophage host range assay: A bacteriophage host range assay can be used to determine which hosts or host cell groups (e.g., bacterial cells) are susceptible to a given bacteriophage in vitro. In some examples, a bacteriophage host range assay is performed using one or more microorganisms isolated from a sample or samples and one or more bacteriophages present in an environmental sample. For example, bacteria can be collected from one or more skin samples, and optionally a portion of the collected bacteria can be sequenced to identify the type of bacteria (e.g., bacterial species or variant). The collected bacteria can be cultured (e.g., on an agar plate), distributed on a phage host range assay dish, and contacted with a viral fraction from an environmental source that may contain one or more bacteriophages (e.g., lytic bacteriophages). Optionally, the collected bacteria can be pre-sorted or processed so that only one strain type or class of strain types is present per dish of the phage host range assay. The bacteria-bacteriophage mixture in the phage host range assay may optionally be cultured. Bacteriophages that grow on or in the collected and cultured bacteria and / or lyse or prevent the growth of the cultured bacteria in the assay may be isolated, optionally propagated, and stored as part of a bacteriophage library. Alternatively or additionally, the bacteriophages may be sequenced. Such sequencing data can then be used to associate each of the isolated bacteriophages with a particular host organism. Thus, such an assay may be useful to determine the identity and genome sequence of a bacteriophage that can remove one or more microorganisms from a sample. Since the identity of one or more microorganisms is also known (e.g., from sequencing), the host range of each bacteriophage can be determined. Thus, for a given target bacterial strain, an appropriate bacteriophage can be selected for inclusion in a formulation designed to lyse the target bacterial strain.

[0050] FIG. 1 shows an exemplary plot of data that can be obtained from a bacteriophage host range assay. The x-axis represents the type or combination of bacteriophages, and the y-axis represents the bacterial strain. In such examples, each grid of the array can represent one or more microorganisms (e.g., bacteria) that can be found in one or more skin samples combined with one or more bacteriophages. Each grid across the array can represent the same type of microorganism (e.g., bacterial strain), or the grids can represent different types of microorganisms (e.g., bacterial strains). In some examples, some of the grids may represent the same type of microorganism and other grids may represent different types of microorganisms. Similarly, the bacteriophages displayed in each grid of the array can be the same or different, or some of the grids may represent the same bacteriophage and others represent different bacteriophages. In some examples, each grid represents a different bacteria-bacteriophage combination. For example, each grid may represent the growth (or lack thereof) of a single microbial (e.g., bacterial) type (e.g., strain) and a single bacteriophage type or combination thereof. In the plot, the density or intensity of each grid represents the increased killing or growth inhibition of the bacterial strain from the bacteriophage type or combination of bacteriophages. In some examples, one or more grids of an assay may represent multiple bacteriophages, which may be useful for determining the susceptibility of a bacterial type or strain to a combination or mixture of bacteriophages. Host range data may be useful for determining combinations of bacteriophages (e.g., lytic bacteriophages) that are effective in lysing or preventing regrowth of a microbiome subtype (e.g., target bacterial strain), which may help prevent bacterial resistance. In some instances, it may be beneficial to avoid lysis of certain strains of "helpful" bacteria (e.g., bacteria associated with a low risk of skin conditions, bacteria that prevent the overgrowth of other bacterial types, bacteria that prevent infection from other microorganisms, certain Staphylococcus epidermidis, C. granulosum, etc.).Therefore, understanding the host range can be useful to generate bacteriophage combinations that selectively avoid lysis of useful bacterial strains.

[0051] Bacteriophage formulations: In some embodiments, methods for generating bacteriophage formulations or mixtures are also provided herein. The bacteriophage formulations or mixtures may include at least one bacteriophage capable of lysing a microorganism (e.g., bacteria) or inhibiting the growth of a microorganism, such as a microorganism found in a biological sample obtained from a subject (e.g., skin, gut, mouth), and in some examples may be useful for treating a health condition (e.g., skin condition or disorder). The method may include determining or identifying the microorganism in the sample (e.g., identifying the host bacteria) and generating a bacteriophage mixture or formulation that includes at least one bacteriophage capable of infecting the microorganism. The bacteriophage mixture or formulation may be administered to the subject from whom the sample was obtained. Thus, the methods provided herein may be useful for generating individualized formulations or mixtures for treating a health condition of an individual. In some examples, the bacteriophage mixture or formulation includes two different bacteriophages, which may have the same or different host ranges. Such combinations of bacteriophages with designated host ranges can help prevent bacterial or microbial resistance.

[0052] FIG. 2 shows exemplary data of bacterial growth in response to infection with a pair of bacteriophages. Each panel represents the growth curve of a bacterial strain following infection with a pair of T4-like bacteriophages. Curves with lighter hues indicate bacterial regrowth following infection, while darker curves indicate little or no bacterial growth. Bacteriophage pairs that result in little or no bacterial regrowth may be candidates for inclusion in a bacteriophage mixture or formulation for treating skin conditions associated with the bacterial strain.

[0053] Bacteriophage selection: Bacteriophages can be selected for inclusion in a mixture or formulation based on any useful characteristics. Characteristics that can be used for bacteriophage selection include, in non-limiting examples, non-lysogenic or non-integrated in the host genome, host range (e.g., specific to the host organism and with minimal off-target binding), and stability in a given formulation. In some examples, combinations of bacteriophages may be selected for a specified microbiome type or host range (e.g., to target a given bacterial strain, such as Propionibacterium acnes), which can be combined to help prevent bacterial resistance.

[0054] Formulations: The present disclosure also provides formulations for administering the bacteriophage or mixture of bacteriophages, for example, to a subject. The formulations may include cosmetic formulations of skin care formulations. The formulations may include any useful ingredient or composition, such as an excipient configured to stabilize one or more bacteriophages (e.g., maintain the percentage of viability of one or more bacteriophages for a period of time, e.g., at least one week). The excipients may include substances for bulking solid, liquid, or gel formulations containing one or more bacteriophages. In some examples, the substances may confer therapeutic enhancements to one or more bacteriophages, for example, by enhancing solubility, decreasing or increasing dissolution, enhancing stability, increasing penetration into the skin or a portion thereof (e.g., the stratum corneum), increasing bacteriophage activity, etc. In some examples, the excipients may include one or more liposomes or lipophilic moieties (e.g., micelles, vesicles) that may encapsulate one or more bacteriophages. The excipient or excipient substance can be used to change the properties of the composition, such as viscosity. The substance can be used to change the properties of the therapeutic agent, such as bioavailability, absorption, hydrophilicity, hydrophobicity, pharmacokinetics, etc. The excipient can include binders, anti-adhesives, coatings, disintegrants, glidants (e.g., silica gel, talc, magnesium carbonate), lubricants, preservatives, adsorbents, sweeteners, vehicles, or combinations thereof. For example, the excipient can include powders, minerals, metals, sugars (e.g., sugars or polysaccharides), sugar alcohols, naturally occurring polymers (e.g., cellulose, methylcellulose), synthetic polymers (e.g., polyethylene glycol or polyvinylpyrrolidone), alcohols, thickeners, starches, polymers (e.g., lipids, proteins, carbohydrates, nucleic acid molecules), etc.

[0055] The formulation may include additional ingredients for treating skin conditions (e.g., aging, acne). For example, the formulation may include one or more anti-aging ingredients, including, but not limited to, retinoids (vitamin A derivatives), niacinamide (vitamin B3), ascorbic acid (vitamin C), skin active peptides, proteins or peptides (e.g., collagen, hyaluronic acid, or derivatives thereof), plant growth factors, such as kinetin, ubiquinone (coenzyme Q10), and the like. Alternatively or additionally, the formulation may include one or more anti-inflammatory agents, such as antihistamines, salicylates, and the like. In some examples, the formulation may include one or more anti-acne agents, such as benzoyl peroxide, salicylic acid, probiotics, antibiotics, antifungals.

[0056] Skin conditions: Bacteriophage mixtures or formulations can be used to treat skin conditions that may be caused by or associated with a particular microbiome subtype (e.g., a particular microorganism, such as a bacterial strain, or combination of microorganisms). For example, the formulations described herein can be used to treat acne, inflammation, redness, eczema, rosacea, enlarged hair follicle pore size, rough skin texture, increased transepidermal water loss, dry skin, skin discoloration (e.g., hyperpigmentation), and uneven elasticity of the skin or a portion thereof (stratum corneum, dermis, etc.). The formulations can include one or more bacteriophages that can lyse or inhibit the growth of one or more microorganisms associated with a skin condition, such as Propionibacterium acnes or Staphylococcus aureus, which may be associated with acne.

[0057] Microbial variants: As described herein, skin samples may contain one or more microorganisms associated with skin conditions (e.g., acne, eczema, redness, etc.). Thus, bacteriophage mixtures and formulations may target any species or variant of skin microorganisms that may be associated with a skin condition. For example, one or more bacteriophages may target bacteria, such as bacteria from the phyla Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. The one or more microorganisms may, in some examples, include Cutibacterium bacteria (e.g., Propionibacterium acnes, C. namnatense, C. avidum), Staphylococcus bacteria (e.g., Staphylococcus aureus, Staphylococcus epidermidis, S. warneri, Streptococcus pyogenes, S. mitis), Corynebacterium bacteria, Acinetobacter bacteria (e.g., A. johnsonii), Pseudomonas bacteria (e.g., Pseudomonas aeruginosa), other bacteria, or combinations thereof. In some examples, the one or more bacteriophages can target fungal microorganisms, including, but not limited to, yeasts such as Candida albicans, Rhodotorula rubra, Torulopsis spp. and Trichosporon cutaneum, dermatophytes such as Microsporum gypseum and T. rubrum, and non-dermatophyte fungi such as Rhizopus stolonifer, Trichosporon cutaneum, Fusarium, Scopulariopsis brevacaulis, Alternaria alternata, Paecilomyces, Aspergillus flavus, and Penicillium. One or more bacteriophages may target a combination of microorganisms.Such combinations can be determined, for example, using a host range assay, as described elsewhere herein.

[0058] Kits: Also provided herein are kits for obtaining samples (e.g., skin samples) from a subject. Such kits may include, for example, sample collectors such as tape patches or strips, swabs, wipes, etc., or any other useful collectors. The kits may include vessels or containers for holding samples or sample collectors, and optionally reagents such as stabilizers, buffers, preservatives, fixatives, pH balancing reagents, etc. The kits may further include instructions for sample collection, storage, transport, processing, etc.

[0059] Systems: In some embodiments, disclosed herein are systems for determining a microbiome type from a sample (e.g., a skin sample), the system including a sequencer configured to perform shotgun metagenomic sequencing and one or more processors configured to process the set of sequence reads to determine the microbiome type.

[0060] Computer Systems The present disclosure provides a computer system programmed to carry out the method of the present disclosure. Figure 3 shows a computer system 301 programmed or otherwise configured to analyze or process sequence reads from shotgun metagenomic sequencing. The computer system 301 can regulate various aspects of the processing of the sequence reads of the present disclosure, such as, for example, performing clustering analysis, performing transformation, normalizing sequence reads to a standard or negative control, etc. The computer system 301 can be a user's electronic device, or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0061] The computer system 301 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 305, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 301 also includes memory or memory locations 310 (e.g., random access memory, read-only memory, flash memory), electronic storage 315 (e.g., hard disk), communication interface 320 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 325, such as cache, other memory, data storage, and / or electronic display adapters. The memory 310, storage 315, interface 320, and peripheral devices 325 communicate with the CPU 305 via a communication bus (solid lines), such as a motherboard. The storage 315 may be a data storage device (or data repository) for storing data. The computer system 301 may be operatively coupled to a computer network ("network") 330 with the aid of the communication interface 320. The network 330 may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 330 may be a telecommunications and / or data network in some cases. The network 330 may include one or more computer servers, which may enable distributed computing such as cloud computing. The network 330 may implement a peer-to-peer network in some cases, which may enable devices coupled to the computer system 301 to operate as clients or servers with the help of the computer system 301.

[0062] The CPU 305 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 310. The instructions may be directed to the CPU 305, which may then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 305 may include fetch, decode, execute, and writeback.

[0063] The CPU 305 may be part of a circuit, such as an integrated circuit. One or more other components of the system 301 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0064] Storage device 315 may store files such as drivers, libraries, and saved programs. Storage device 315 may store user data, e.g., user preferences and user programs. Computer system 301 may include one or more additional data storage devices, possibly located outside computer system 301, e.g., on a remote server in communication with computer system 301 via an intranet or the Internet.

[0065] Computer system 301 can communicate with one or more remote computer systems via network 330. For example, computer system 301 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., portable PC), a slate or tablet PC (e.g., Apple® iPad, Samsung® Galaxy Tab), a phone, a smartphone (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or a personal digital assistant. A user can access computer system 301 via network 330.

[0066] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored in electronic storage locations of the computer system 301, such as memory 310 or electronic storage 315. The machine executable or machine readable code may be provided in the form of software. In use, the code may be executed by the processor 305. In some cases, the code may be retrieved from storage 315 and stored in memory 310 for easy access by the processor 305. In some situations, the electronic storage 315 may be omitted and the machine executable instructions are stored in memory 310.

[0067] The code may be pre-compiled and configured for use on a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be provided in a programming language, which may be selected so that the code can be executed in a pre-compiled or as-compiled manner.

[0068] Aspects of the systems and methods provided herein, such as computer system 301, can be embodied in programming. Various aspects of the technology can be considered as a "product" or "article of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried on or embodied in some type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, a processor, or associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage at any time for software programming. All or part of the software can be communicated over the Internet or various other telecommunications networks. Such communication can, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, another type of medium that may bear software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and across various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0069] Thus, a machine-readable medium such as a computer executable code can take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include any of the storage devices such as optical or magnetic disks, any computer that can be used to implement, for example, the databases shown in the drawings. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and optical fibers, including the wires that comprise a bus within a computer system. Carrier wave transmission media can take the form of electric or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave carrying data or instructions, a cable or link carrying such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media can be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0070] The computer system 301 includes an electronic display 335 that can include or communicate with a user interface (UI) 340 for providing, for example, sequence read alignment, binning, clustering, normalization to a standard or control, etc. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0071] The disclosed methods and systems can be implemented by one or more algorithms. The algorithms can be implemented by software upon execution by the central processing unit 305. The algorithms can, for example, process sequence reads, such as aligning, binning, assembling, normalizing, clustering, transforming, taxonomic profiling, and the like.

[0072] [Example] [Prophetic Example 1] Phage host range assay to identify bacteriophages that infect Propionibacterium acnes Bacteriophage host range assays can be used to determine which hosts or host cell populations (e.g., bacterial cells) are susceptible to a given bacteriophage in vitro. In some instances, it may be useful to identify bacteriophages that infect P. acnes bacteria. To do so, P. acnes strains can be cultured, distributed onto a phage host range assay plate or plates, and contacted with bacteriophages. The bacteria-bacteriophage mixtures in the phage host range assay (e.g., from each assay in a series of assays) can optionally be cultured or incubated. Sequencing (e.g., metagenomic shotgun sequencing) can be performed on the bacteria-bacteriophage mixtures to determine the identity of the bacteriophages and / or bacteria present in each mixture.

[0073] Sequencing of DNA extracted from bacteriophage plaques on a host range assay plate (e.g., shotgun metagenomic sequencing) can be used to identify one or more bacteriophages. Sequencing of DNA extracted from bacteria growing on the plate (e.g., shotgun metagenomic sequencing) can be used to identify P. acnes strains. In some examples, DNA is extracted from both bacteriophage plaques and bacteria growing on the plate. In some examples, it may be beneficial to include an internal DNA standard, for example, as a positive control or to normalize sequence counts. For example, a DNA standard of known sequence can be spiked into a sample (e.g., a sample containing DNA extracted from bacteriophages or bacteria) prior to sequencing. After sequencing, the presence of a positive control read can help verify that the sequencing was performed accurately. In some examples, an internal DNA standard can be used to normalize or quantify sequencing counts. For example, an internal DNA standard can be spiked into a sample at a known concentration, and subsequent sequencing read counts can be normalized based on the starting known concentration to determine the relative or absolute amount of each of the resulting sequences.

[0074] Sequencing data (e.g., sequence reads) can be further processed or analyzed as described herein, for example, to identify genetic elements of bacteriophages related to host range patterns. For example, one or more clustering analyses can be performed to determine sequence segments common to bacteriophages that can infect a given P. acnes strain. Thus, the determined sequence segments can be useful for identifying bacteriophages or bacteriophage types that can lyse P. acnes.

[0075] [Prophetic Example 2] Determining the type or subtype of the skin microbiome The methods, systems and kits described herein may be useful in determining the type or subtype of the microbiome from a subject, and administering a composition comprising a bacteriophage that can treat a condition in the subject.

[0076] In one example, a skin sample can be obtained from a subject (e.g., using a kit described herein). The subject may have or be suspected of having a skin condition (e.g., acne). The skin sample can be further processed, for example, to extract DNA from the skin sample. An internal standard can be added to the extracted DNA. The extracted DNA can be subjected to sequencing (e.g., metagenomic shotgun sequencing), which can be used to determine the type or subtype of the subject's microbiome. For example, the sequenced DNA can indicate the presence of one or more Propionibacterium acnes bacteria, or other bacterial strains that may be associated with the skin condition (e.g., acne). The relative abundance of each bacteria can be determined with reference to sequence reads from the internal standard. A composition comprising one or more bacteriophages can then be administered to the subject. The one or more bacteriophages can include bacteriophages that can infect and lyse bacterial strains present in the skin sample. For example, in the case of a skin sample containing one or more P. acnes bacteria, one or more bacteriophages capable of lysing the P. acnes bacteria can be administered to a subject, which can be useful for treating a skin condition (e.g., acne). In some examples, the composition can include two or more bacteriophages capable of lysing the same P. acnes strain, which can help reduce bacterial resistance. In some examples, the composition can include two or more bacteriophages capable of lysing different P. acnes strains. In some examples, a bacteriophage cocktail can be administered to target one or more bacterial strains associated with a skin condition. One or more bacteriophages can specifically target a bacterial strain associated with a skin condition (e.g., acne) and not target other bacteria not associated with the skin condition (e.g., C. granulosum). Administration of such a composition containing one or more bacteriophages to a subject can be useful for treating a skin condition.

[0077] [Example 3] Bacteriophage host range assays against P. acnes, C. namnatense, and C. granulosum As described herein, bacteriophage host range assays can be used to determine which hosts or host cell populations (eg, bacterial cells) are susceptible to a given bacteriophage in vitro.

[0078] An example of a bacteriophage host range assay protocol is provided as follows.

[0079] 1. Prepare an agar-layered Petri dish by combining 10 mL of molten top agar with less than 500 µL of putative host bacteria and pouring on top of a solid base of 1.5% agar medium in a 12 square cm Petri dish.

[0080] 2. Serially dilute the phage stock in SM buffer.

[0081] 3.10 -1 , then 10 -4 Pipette 5-10 microliters (µL) of the phage stock of the dilution (or any other dilution selected) onto an organized grid on an agar-overlaid Petri dish.

[0082] 4. Repeat steps 2-3 with different phage stocks.

[0083] 5. Absorb a 10 μL drop of phage from each phage stock dilution onto the top agar.

[0084] 6. Place the agar overlay plate in an incubator under growth conditions appropriate for the host bacteria.

[0085] 7. After the bacteria have formed a visible lawn during incubation, the plates are evaluated for the occurrence of plaque formation where phage dilutions are pipetted onto the agar-overlaid Petri dishes.

[0086] 8. If plaque formation is visible in one or both of the phage spots, score the phage host range accordingly.

[0087] Figure 4 shows exemplary data from a bacteriophage host range assay generated using the protocol described above. The y-axis represents 96 different bacteriophages and the x-axis represents 13 different bacterial strains. The bacterial strains on the x-axis represent, from left to right, strain numbers 100047 (C. namnatense strain), 100048 (P. acnes subsp. defendens strain), 100057 (C. avidum strain), 100069 (C. granulosum strain), 100085 (P. acnes strain), 100088 (P. acnes strain), 100111 (P. acnes strain), 100119 (P. acnes strain), 100124 (P. acnes strain), 100126 (P. acnes strain), 100132 (P. acnes strain), 100144 (P. acnes strain), and 100146 (P. acnes strain). Each grid of the array represents a single assay of a bacterial strain (x-axis) contacted with a bacteriophage (y-axis). In the plot, the density or intensity of each grid represents the killing or growth inhibition of the bacterial strain from the bacteriophage type. A white square indicates that the bacteriophage does not infect the bacterial strain. A black square indicates that the bacteriophage infects the bacterial strain.

[0088] As can be seen in Figure 4, all but 11 bacteriophages do not infect C namnatense strains (row 1), and all but 3 bacteriophages do not infect C granulosum strains (row 4). Many of the phages infect multiple P. acnes strains. Bacteriophages with useful properties (e.g., infecting most or all P. acnes strains and C. namnatense, but not C. granulosum) can be selected for inclusion in formulations to treat acne, for example.

[0089] Overall, the host range data may be useful to determine bacteriophages that are effective in lysing or preventing regrowth of a particular bacterial strain. Additionally, the host range data may be useful to select combinations of bacteriophages for lysing or preventing regrowth of one or more bacterial strains. For example, the host range data may be useful to determine two or more bacteriophages that can lyse the same or different P. acnes strains (e.g., bacteriophages 1-13 can all lyse P. acnes strain 100048), and such combinations of bacteriophages may be useful to prevent the emergence of variant strains that are resistant to one of the bacteriophages. Alternatively or additionally, the two or more bacteriophages may have other favorable properties, such as not infecting or lysing other bacterial types, such as C. granulosum. In another example, the host range data may be useful to determine two or more bacteriophages that, alone or in combination, infect all bacterial species in a group. For example, the combination of bacteriophages 13 and 58 can lyse all tested P. acnes strains, and the combination of 13, 58 and 87 can lyse all tested P. acnes and C. namnatense strains. Particularly useful combinations of bacteriophages include bacteriophages 2, 3, 7, 8, 9, 11, 67, 69, 85, 87 and 96.

[0090] To further characterize the bacteriophage and / or bacterial strain, one can sequence the DNA extracted from the bacteriophage plaques, which may contain one or more DNA standards (as described elsewhere herein). An exemplary protocol for preparing bacteriophage plaques for sequencing is as follows.

[0091] 1. Prepare phage stocks in broth medium.

[0092] 1. Lysing susceptible bacterial cultures by adding phages during the exponential growth phase of bacteria 2. Remove most of the bacterial biomass from the lysate by centrifugation at low speed and retaining the supernatant containing the phage 3. Filter the clarified lysate through a 0.2um filter to sterilize the phage stock 2. Extract DNA from phage stocks.

[0093] 1. Remove exogenous DNA by incubating phage stocks with DNAse 2. Remove DNAse and digest phage capsids by incubation with proteinase K and SDS 3. Purify DNA by loading onto a commercial silica-column DNA clean-up kit and washing with 80% ethanol 3. Prepare a DNA library from the purified DNA using one of the commercially available DNA sequencing library preparation kits such as the Illumina Nextera XT or TruSeq kits.

[0094] 4. Assay the quality of the DNA library and load onto the sequencing instrument.

[0095] 5. Assemble the DNA sequencing reads into a single contig representing the phage genome.

[0096] The sequencing data (e.g., sequence reads) can be further processed or analyzed as described herein, for example, to identify genetic elements of the bacteriophage that are associated with host range patterns. For example, one or more clustering analyses can be performed to determine sequence segments common to bacteriophages that can infect a given Cutibacterium strain. The determined sequence segments can thus be useful for identifying bacteriophages or bacteriophage types that can lyse a selected Cutibacterium strain.

[0097] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in the practice of the present invention. It is therefore contemplated that the present invention encompasses any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A bacteriophage or fungal virus preparation capable of lysing microorganisms of a population of microorganisms for use in treating a skin condition in a subject, said use comprising: (a) detecting the population of microorganisms in a skin sample obtained from the subject, comprising performing shotgun metagenomic sequencing of nucleic acid molecules extracted from the population of microorganisms, thereby generating a set of sequence reads; (b) processing the set of sequence reads to determine a microbiome type of the microbial population; and (c) administering the bacteriophage or fungal virus preparation to the subject to lyse the microorganism identified from the set of sequence reads. formulation.

2. The formulation of claim 1 , wherein the set of sequence reads consists of from 0.1M to 100M sequence reads.

3. 3. The formulation of claim 1 or 2, wherein the population of microorganisms comprises bacteria, fungi, mites or viruses.

4. 4. The formulation of any one of claims 1 to 3, wherein the population of microorganisms comprises one or more microorganisms associated with acne, inflammation, redness, eczema, rosacea, enlarged hair follicle pore size, rough skin texture, increased transepidermal water loss, skin discoloration, unequal elasticity of the stratum corneum and underlying dermis, or aging skin.

5. The formulation of any one of claims 1 to 4, wherein the population of microorganisms comprises Propionibacterium acnes bacteria, Staphylococcus aureus bacteria, Corynebacterium sp. bacteria, or Malassezia yeast.

6. The formulation of any one of claims 1 to 5, wherein the detecting further comprises: (1) isolating deoxyribonucleic acid (DNA) from the skin sample; and (2) removing human DNA from the DNA isolated in (1).

7. The formulation of claim 6, wherein the use comprises adding an internal DNA standard to the isolated DNA, the internal DNA standard comprising synthetic DNA or naturally occurring DNA.

8. 8. The formulation of any one of claims 1-7, wherein the processing comprises (i) identifying one or more background sequence reads by shotgun genomic sequencing of deoxyribonucleic acid (DNA) isolated from a negative control sample, and (ii) eliminating the one or more background sequence reads from the set of sequence reads.

9. The formulation of any one of claims 1 to 8, wherein the processing comprises normalizing counts of the set of sequence reads to generate a set of normalized sequence reads.

10. The formulation of claim 9, wherein the processing comprises (i) clustering the set of normalized sequence reads and / or (ii) comparing the set of normalized sequence reads to a database, the database comprising sequences of a plurality of microorganisms isolated from other skin samples from other subjects, or sequences of mobile genetic elements comprising one or more members of the group consisting of plasmids, prophages and transposons.

11. 11. The bacteriophage or fungal virus formulation of any one of claims 1 to 10, wherein the bacteriophage or fungal virus formulation comprises a first bacteriophage and a second bacteriophage, wherein the first bacteriophage and the second bacteriophage (i) are capable of lysing one or more microorganisms of the population of microorganisms, or (ii) are lytic bacteriophages.

12. 12. The formulation of claim 11, wherein the first bacteriophage and the second bacteriophage have different host ranges, the different host ranges comprising (i) at least two Propionibacterium acnes strains, or (ii) at least one Propionibacterium acnes strain and at least one C. namnatense strain.

13. 13. The formulation of claim 12, wherein the different host range does not include a C. granulosum strain.

14. 12. The formulation of claim 11, wherein the first bacteriophage and the second bacteriophage individually or collectively prevent resistance or regrowth of the one or more microorganisms in vitro.

15. 15. The formulation of any one of claims 11 to 14, wherein the bacteriophage or fungal virus formulation is part of a cosmetic formulation, the cosmetic formulation comprising one or more liposomes containing the first bacteriophage and the second bacteriophage.

16. 16. The formulation of claim 15, wherein the cosmetic formulation further comprises an anti-aging ingredient.

17. 1. A method for determining a microbiome type, comprising: (a) combining DNA isolated from a subject's skin sample with an internal DNA standard to form a DNA mixture; (b) performing shotgun metagenomic sequencing of the DNA mixture to generate a set of sequence reads; and (c) processing the set of sequence reads to determine the type of the microbiome.

18. 18. The method of claim 17, wherein the subject has acne, redness, eczema, rosacea or aging skin.

19. 18. The method of claim 17, wherein the DNA isolated from the skin sample contains less than 10% human DNA.

20. 18. The method of Claim 17, wherein said processing comprises transforming the set of sequence reads to generate transformed sequence reads, and clustering the transformed sequence reads.

21. 21. The method of any one of claims 17 to 20, wherein the processing comprises (i) normalizing counts of a subset of the set of sequence reads to another count of a set of sequence reads from the internal DNA standard, and / or (ii) comparing the plurality of sequence reads to a set of sequence reads of a negative control sample, the negative control sample being open to air for a short period of time together with the skin sample and processed in parallel to the skin sample.

22. The method of any one of claims 17 to 21, wherein the processing comprises detecting one or more subtypes of Propionibacterium acnes or Staphylococcus aureus.

23. (a) contacting (i) a sample from an environmental source with (ii) one or more cultured microorganisms from a skin sample; and (b) isolating a lytic bacteriophage capable of lysing the one or more cultured microorganisms; and (c) sequencing the nucleic acid isolated from the lytic bacteriophage; method.

24. 24. The method of claim 23, wherein the environmental source is sewage or a soil suspension.

25. 25. The method of claim 23 or 24, further comprising identifying the one or more cultured microorganisms by a method comprising DNA sequencing.

26. 26. The method of any one of claims 23 to 25, wherein the one or more cultured microorganisms comprise a Propionibacterium acnes strain, a Staphylococcus aureus strain, or a Corynebacterium strain.

27. 27. The method of any one of claims 23 to 26, wherein the skin sample comprises a skin swab from skin affected by acne, inflammation, redness, eczema, rosacea, or ageing.

28. 28. The method of any one of claims 23 to 27, further comprising determining the host range of the lytic bacteriophage, wherein the host range does not include beneficial bacteria associated with a low risk of a skin condition.

29. 29. The method of any one of claims 23 to 28, further comprising constructing a bacteriophage library comprising the lytic bacteriophage, wherein the bacteriophage library comprises a plurality of lytic bacteriophages capable of lysing the one or more cultured microorganisms.

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