Method for identifying microbial strains

Abstract

Described herein are methods for identifying microbial strains from a sample.

Description

Attorney Docket No.: 273089 / KBS-007WO / 576924METHOD FOR IDENTIFYING MICROBIAL STRAINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 716,534, filed November 5, 2024, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] This invention relates to methods for identifying microbial strains from a sample.BACKGROUND

[0003] The field of microbiome research is pivotal in understanding and manipulating the complex communities of microorganisms that reside in various environments, including the human body. This area of study holds promise for the development of microbiome therapeutics, a novel and exciting frontier in medicine. However, the success of these therapeutic interventions relies heavily on the ability to isolate and culture specific microbial strains from natural samples, such as fecal material. This is crucial for replacing fecal microbiota transplantation (FMT) with synthetic communities that offer a more controlled and safer alternative. In addition to human therapeutic discovery, it is important to achieve targeted bacterial strain isolation from a complex microbial starting sample.SUMMARY

[0004] One aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) diluting the sample to obtain a diluted sample;(iii) dispersing the diluted sample into a multi-well plate; and(iv) analyzing the wells with a screen to identify microbial strains.

[0005] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate to obtain live cultures;(vi) optionally, identifying wells of interest with a first screen or growth and consolidating the wells of interest into at least one second multi-well plate; and(vii.A) analyzing the first multi-well plate or second multi-well plate with a first screen to identify microbial strains;(vii.B) analyzing the first multi -well plate or second multi -well plate with a second screen to identify microbial strains; and / or(vii.C) storing the live cultures of the first or second multi -well plate.

[0006] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate;(v.A) identifying wells of interest, wherein the wells of interest are wells with growth;(vi) pooling wells of interest to obtain a pooled sample, wherein the pooling comprises removing a portion of the wells of interest;(vii) plating the pooled sample on an array;(viii) identifying a pool of interest with a first screen;(ix) isolating the pool of interest and performing steps (iii)-(v) and (vii)-(viii) on the pool of interest;(x) performing a second screen to identify a culture of interest from the pool of interest;262735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(xi) isolating the culture of interest and performing a third screen until a microbial strain of interest is identified.

[0007] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies or biomass;(iii.A) picking live colonies and depositing the live colonies on an array; or(iii.B) picking live colonies and depositing the live colonies on a container for fixation; then transferring the fixed colonies to an array; or(iii.C) stamping an array onto the live colonies directly;(iii.C. l) optionally, fixing the colonies on the array;(iv) staining the array of any of steps (iii.A)-(iii.C);(v) performing a screen on the array of any of steps (iii.A)-(iii.C) to identify microbial strains of interest;(vi) optionally, repeating steps (iii.A)-(v); and(vii) optionally, re-culturing the microbial strain of interest from the plate in step (ii) and repeat steps (iii)-(v).

[0008] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies;(iii) stamping a first array onto the live colonies directly;(iv) stamping a second array onto the first array;(v) generating at least one replica plate by stamping the first array onto a new plate;(vi) fixing and staining the second array;(vii) performing a screen on the second array to identify microbial strains of interest;(viii) optionally, repeating steps (iii)-(v); and(ix) optionally, re-culturing the microbial strain of interest and repeat steps (ii)-(v).

[0009] Other aspects, embodiments, and features as disclosed herein will be apparent from the following description, the drawings, and the claims.362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A shows a schematic of HiPR-ID screening workflow.

[0011] FIG. IB shows a HiPR-ID screening example.

[0012] FIG. 2A shows a schematic of stages of a HiPR-ID-enabled isolation campaign.

[0013] FIG. 2B shows a HiPR-ID baseline example.

[0014] FIG. 3A-3F shows various HiPR-ID Representative Results. FIG. 3A HiPR-ID identifies pure cultures. FIG. 3B HiPR-ID identifies low abundance taxa in mixed cultures. FIG. 3C HiPR-ID and imaging can give more information than MALDI. FIG. 3D HiPR-ID can give a result with low biomass. FIG. 3E HiPR-ID can enable high-throughput screening. FIG. 3F Morphology information from HiPR-ID workflow can guide isolation.

[0015] FIG. 4A shows a schematic of pooling samples to increase HiPR-ID throughput.

[0016] FIG. 4B shows a HiPR-ID example on pooled samples.

[0017] FIG. 5A shows a schematic of HiPR-ID on colonies.

[0018] FIG. 5B shows an example of HiPR-ID on colonies.

[0019] FIG. 5C shows an example of HiPR-ID on solid media cultures.

[0020] FIG. 6A shows a schematic of HiPR-ID on colonies with replica plating.

[0021] FIG. 6B shows an example of fixation on agar and HiPR-ID.

[0022] FIG. 7A shows a schematic of proposed two-stage workflow, where a first array is used to generate both the replica plate and the array with colonies for assay / imaging.

[0023] FIG. 7B shows a master plate containing inoculum of S. epidermidis, E. coH, C. freundii, and mix. The box indicates region where slide shown in FIG. 7C covered.

[0024] FIG. 7C shows a replica plate generated from master plate in FIG. 7B.

[0025] FIG. 7D shows a widefield DAPI tilescan of slide with colonies from plate shown in FIG. 7B, which had been fixed and processed with HiPR-FISH. Scan visualized regions of colony transfer. Indicated colonies were imaged at 63X with confocal spectral imaging.

[0026] FIG. 7E shows HiPR-ID classification results showing S. epidermidis classification (left) and mixed taxa (right), matching the inoculum in those regions.INCORPORATION BY REFERENCE

[0027] 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, or462735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 patent application was specifically and individually indicated to be incorporated by reference.

[0028] In particular, the entire contents of each of the following patent applications are incorporated herein by reference in their entireties: PCT Application Nos.: PCT / US2019 / 021088; PCT / US2022 / 080355; and PCT / US2023 / 062917; and U.S. Application Nos.: 16 / 978,891; 18 / 058,171; and 18 / 171,850; and U.S. Provisional Application No. : 63 / 720,519.DETAILED DESCRIPTION

[0029] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present methods and compositions are described below in various levels of detail in order to provide a substantial understanding of the present disclosure.

[0030] Definitions

[0031] Where values are described as ranges, endpoints are included. Furthermore, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0032] “5’ -end” and “3 ’-end” refers to the directionality, e.g., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0033] The term “about,” as used herein, refers to + / - 10% of a recited value.

[0034] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two nucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first nucleotide is complementary to a second nucleotide if the nucleotide sequence of the first nucleotide is substantially identical to the nucleotide sequence of the nucleotide binding partner of the second nucleotide, or if the first nucleotide can hybridize to the second nucleotide under stringent hybridization conditions. Thus, the nucleotide whose sequence is 5 '-T AT AC-3' is complementary to a nucleotide whose sequence is 5'-GTATA-3'.

[0035] “Nucleotides,” “Nucleic acids,” “polynucleotide” or “oligonucleotide” refer to a polymeric-form of DNA and / or RNA (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides.562735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924As used herein, the term “nucleotides” includes double- and single-stranded DNA, as well as double- and single-stranded RNA; it also includes modified and unmodified forms of a nucleotide (modifications to and of a nucleotide, for example, can include methylation, phosphorylation, and / or capping). In some embodiments, a nucleotide can be one of the following: a gene or gene fragment; genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA (tRNA); ribosomal RNA (rRNA); ribozyme; cDNA; recombinant nucleotide; branched nucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; any DNA described herein, any RNA described herein, primer or amplified copy of any of the foregoing.

[0036] In some embodiments, nucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of nucleotides can also be referenced to by their 5’- or 3’- end or terminus, which indicates the directionality of the nucleotide sequence. Adjacent nucleotides in a single-strand of nucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons. However, different internucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the nucleotide sequence, which may be called the 5’ and 3’ ends or termini. The 5’ and 3’ ends can also be called the phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term “nucleotides” also refers to both double- and single-stranded molecules.

[0037] In some embodiments, nucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with non-natural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the nucleotide sequence.

[0038] In some embodiments, the sequence of nucleotides can be interrupted by nonnucleotide components. One or more ends of the nucleotides can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other nucleotides.

[0039] In some embodiments, nucleotides can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be present, for example, as a natural replacement for thymine when the nucleotide is RNA. Uracil can662735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of nucleotides or any nucleic acid molecule, including natural and non-natural bases.

[0040] When used in terms of length, for example 20 nt, “nt” refers to nucleotide(s).

[0041] As used herein a “taxon” refers to a group of one or more populations of an organism or organisms. In some embodiments, a “taxon” refers to a phylum, a class, an order, a family, a genus, a species, or a strain. In some embodiments, the disclosure includes providing a list of taxa of microorganisms. In some embodiments, the list of taxa of microorganisms is selected from a list of phyla, a list of classes, a list of orders, a list of families, a list of genera, or a list of species of microorganisms.

[0042] In analysis of a sample, a species can be a target of interest. For example, a species can include a taxonomic species.

[0043] A “pathogen” refers to any agent capable of causing an infection or a disease in a host, i.e., a cell or subject. In some embodiments, a pathogen is a microorganism or microbial fragment. In some embodiments, the pathogen may comprise a whole (infectious) pathogen cell, or a part of the pathogen cell, such as, a cell wall component of a microorganism. In some embodiments, the pathogen comprises a pathogen fragment, a pathogen debris, a pathogen nucleic acid, a pathogen lipoprotein, a pathogen surface glycoprotein, a pathogen membrane component, or a component released from the pathogen. In some embodiments, the pathogen is, is derived from, or is isolated from bacteria, fungus, prokaryote, virus, phage, or a misfolded protein (e.g., a prion). In some embodiments, the pathogen is genetically modified.

[0044] A “microbial strain” refers to any strain derived from a microbial species. A “microbial strain” may also be any strain that can potentially ameliorate a disease phenotype. In some embodiments, a microbial strain is any strain derived from a microbe and which does not cause a disease in a host, i.e., a cell or subject. In some embodiments, a microbial strain produces a therapeutic effect. In some embodiments, a microbial strain is a strain that acts against the pathogen. A microbial strain used in the methods described herein may derive from a healthy cell or subject and / or may contain healthy or desirable microbes. In some embodiments, a microbial strain is selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism. In some embodiments, a genetically modified organism is an organism wherein specific genes have been added, deleted, mutated, driven to high expressions, and / or suppressed to low expression levels.762735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924In some embodiments, the genetic modifications in the genetically modified organism are performed via electroporation, conjugation, transformation, transduction, CRISPR-Cas9 system, TALENs (Transcription Activator-Like Effector Nucleases), homologous recombination, sitespecific recombinases, or bacterial artificial chromosomes. In some embodiments, a microbial strain is a bacterial strain such as a probiotic. The microbial strain of the methods described herein is to provide therapeutic / beneficial effects to the host and therefore does not cause a disease in said host. It is possible that microbial strains may be (or be derived from) strains that do not cause a disease in a normal host, but can cause disease in a subpopulation (e.g., an immunocompromised subject).

[0045] In the event of any term having an inconsistent definition between this application and a referenced document, the term is to be interpreted as defined herein.HiPR-ID

[0046] The field of microbiome research is pivotal in understanding and manipulating the complex communities of microorganisms that reside in various environments, including the human body. This area of study holds promise for the development of microbiome therapeutics, a novel and exciting frontier in medicine. However, the success of these therapeutic interventions relies heavily on the ability to isolate and culture specific microbial strains from natural samples, such as fecal matter. Successful isolation is crucial for replacing fecal microbiota transplantation (FMT) with synthetic communities that offer a more controlled and safer alternative.

[0047] The conventional approach to microbial isolation involves manually picking colonies or cultures and performing 16S rRNA gene sequencing to identify the isolates. The process can be augmented by observing features such as colony morphology and using technologies like Matrix- Assisted Laser Desorption / Ionization (MALDI) spectra analysis. However, this traditional method is notoriously laborious and time-consuming. Moreover, as an isolation campaign continues, the rate of new strain isolation tends to plateau, especially for low-abundance microbes, which are particularly challenging to isolate due to their minimal presence in samples. Viability is also an issue. Certain natural isolates which are difficult to culture may die during isolation while still generating an ID using MALDI / WGS, thus leading to wasted time and effort of further attempts at regrowing.

[0048] Several techniques have arisen over the past several years to facilitate faster and more862735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 automated isolation; these tend to use machine learning and rely on macroscopic imaging of colonies on agar to assist in isolation. However, many microbes generate colonies with similar macroscopic characteristics and thus cannot be differentially identified via macroscopic imaging.

[0049] Addressing the limitations of traditional methods, the application of the Poisson distribution has emerged as an approach to facilitate the isolation of microbial strains from liquid culture. This mathematical model aids in predicting the number of starting microbes needed to generate liquid cultures in well-plates, thus reducing the necessity for single-colony growth. This technique not only streamlines the isolation process but also makes the subsequent cry opreservation and drug formulation steps more efficient.

[0050] The use of MALDI-TOF mass spectrometry offers a quick, same-day identification of species from liquid cultures or colonies, provided that their spectra are already available in databases. The application of this technology marked a significant advancement in the field of culturomics. However, the utility of MALDI-TOF is limited by its dependency on existing spectral libraries. Consequently, many microbial taxa, which are previously uncultured or isolated, remain unidentified. Additionally, certain species do not yield clear results through MALDI-TOF, and mixed culture wells can further complicate the identification process. Throughput is also an issue, as the MALDI chip accommodates 96 samples, thus limiting throughput to only about 96 samples a day. Further, a MALDI result does not necessarily correspond with viability of a sample for further growth.

[0051] Whole genome sequencing (WGS) presents a comprehensive solution for microbial identification but lacks the distinction between DNA from intact, viable cells vs. free DNA. Detection of all DNA, including from free DNA and non-viable cells can confound cultivation efforts. This issue highlights the need for a more accurate match between what is viable and what is culturable, especially important for cultivation of lower abundance, difficult-to-culture strict anaerobes of the human gut. Further, WGS is often prohibitively expensive, especially when thousands of colonies or cultures must be screened to detect microbes of low abundance. These low-abundance microbes, however, cannot be overlooked as they often play crucial roles in the microbiome's overall function and can be essential for maintaining health. Also, a certain amount of biomass is needed to generate a MALDI ID or sufficient material for sequencing; however, many microbes of interest which are difficult to culture may not reach high density during a large- scale isolation campaign and thus not be identified with conventional methods.962735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0052] Accordingly, there is an acute need for new technology to enable identification of microbrial strains from a sample, such as fecal matter.

[0053] In response to these challenges, the present disclosure describes HiPR-ID, a sensitive and specific method for detecting relevant strains. This technique can be applied to a variety of sample formats, including those containing previously uncultured strains. The development of Fluorescence in situ hybridization (FISH), combined with culture independent DNA extraction and 16S amplification, revolutionized microbial ecology by providing direct insight into ‘who is there’. These techniques revealed the gap between what had previously been isolated and what exists in nature. Many known strains have never been isolated and are difficult to detect without sequencing, reducing the likelihood that a rare culture of such a strain would be successfully detected during an isolation effort. While prior FISH approaches allowed for only probing a limited set of taxa simultaneously, the development of HiPR-FISH allowed for simultaneous detection of over 1000 targets. Applying HiPR-FISH to isolation samples can allow the sensitive and specific detection of vast numbers of strains simultaneously what is detected in situ and what grows on petri plates, called the “great plate count anomaly”. HiPR-FISH offers a quicker and more cost- effective alternative to sequencing, thus providing a significant advantage for large-scale screening and identification efforts in microbiome research.

[0054] The identification method described herein presents several key advantages over existing techniques. For example, the high-throughput application of HiPR-FISH in the presently described method allows for quick and robust identification of strains of interest from a cultured microbiome sample by location, in such a way that the location can be re-cultured for further analysis and banking, and requires only limited biomass, allowing the preservation of biomass in the original cultured sample such that that the identified location can be re-cultured for further analysis and banking. While limited prior work exists on using FISH directly on live cells, which could enable further growth without such re-culturing, such fixation-free approaches are limited in application to qualitative work and have not yet been demonstrated to achieve the specificity and sensitivity needed for high-complexity samples and probe panels, particularly for Gram positive cells which require additional permeabilization (Suzan Yilmaz, et al., Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations, The ISME Journal, Volume 4, Issue 10, October 2010, Pages 1352-1356). In comparison, a traditional FISH approach on fixed samples, combined with the HiPR-FISH method of probe design, achieves the1062735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 desired sensitivity and specificity needed to pursue the diversity present in a complex microbial sample..

[0055] Another advantage is over MALDI processing. The manufacturer (Bruker) recommends MALDI testing to be performed on cultures scraped from colonies grown on agar and provides a library for identification of strains that returns a species name when the Mass Spectra matches. Failure to match results in one of two outputs: No ID possible (when there is sufficient biomass, but a novel spectra) or No peaks found (when there is insufficient biomass for detection). The Bruker library is optimized to detect clinical pathogens and model aerobic strains with some facultative anaerobes present in the library. However, many strains derived or helpful in the research of the microbiome are commensal anaerobes that often result in “No ID possible.” While the manufacturer’s recommendation is to use colonies, the present Inventors found that washing and plating liquid cultures from well plates reduces the frequency of “No peaks found” from 30 - 50 % for agar to 5 - 15 % for liquid format. However, for liquid cultures from which MALDI would still yield a “No peaks found” result due to low biomass, an incorrect ID due to a mixed culture, or a no ID found, HiPR-ID can generate the correct ID or be used to gain information about the mixed culture via imaged morphologies and spectral characteristics about the microbes present, which can be used to guide further isolation work.

[0056] High Phylogenetic Resolution microbiome mapping by Fluorescence in situ Hybridization (HiPR-FISH), developed by the Applicant, is a versatile technology that uses binary encoding, spectral imaging, and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. See, for example, Shi, H. et al. “Highly multiplexed spatial mapping of microbial communities.” Nature vol. 588, 7839 (2020): 676-681, PCT Patent Publication WO 2019 / 173555, filed March 7, 2019; PCT Patent Application No. PCT / US2022 / 080355, filed on November 24, 2022, and U.S. Application No. 18 / 058,171, filed on November 24, 2022. The contents of the aforementioned disclosures are each incorporated herein by reference in their entireties.

[0057] HiPR-FISH has been able to distinguish between 1023 unique targets in a single experiment. It has been applied to numerous specimen types including mammalian tissue, food products, biofilms, and cultured epithelial cells with adherent bacteria. The present disclosure provides methods to incorporate this technology for identifying and isolating microbial strains from samples of interest.1162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0058] Accordingly, the present disclosure provides various methods for identifying and isolating microbial strains.

[0059] One aspect of the present disclosure provides HiPR-ID methods for identifying microbial strains. For example, a portion of a sample can be diluted and fixed without culturing, stained with DAPI (4',6-Diamidino-2-phenylindole) or an EUB-338 probe, or a pan-fungal probe, then hybridized with a HiPR-ID probe panel, demonstrating presence of a variety of taxa and the prevalence of taxa which would be hybridized by the probes.

[0060] Another HiPR-ID method can be after designing a probe panel against a source sample,HiPR-FISH can be conducted on the baseline source sample to confirm presence of taxa of interest, to predict their viability and metabolic activity (via rRNA intensity or use of viability stains), and to create a reference database of morphologies and expected associated spectra to be used during later stages of isolation. As a first step of an isolation campaign, HiPR-ID high-throughput screening can be applied to determine appropriate growth conditions that enrich for desired taxa to be isolated, to determine an appropriate level of dilution of the source sample for further culturing, and to screen prepared dilutions for ones containing taxa of interest at higher abundance. As a next stage of isolation, specified dilutions of the source sample and growth conditions can be used to create a large number of cultures which can be screened with HiPR-ID for growth of taxa of interest. If identified cultures are mixed, these can be further diluted, grown and screened with HiPR-ID and these steps can be repeated until the taxa of interest is confirmed to be isolated. Isolates can be preserved and banked with cryoprotectant and characterized by genomic sequencing.

[0061] Accordingly, one aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) diluting the sample to obtain a diluted sample;(iii) dispersing the diluted sample into a multi-well plate; and(iv) analyzing the wells with a screen to identify microbial strains

[0062] Accordingly, one aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) diluting the sample to obtain a diluted sample;1262735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(iii) dispersing the diluted sample into a multi-well plate;(iv) identifying wells of interest; and(v) analyzing the wells of interest with a screen to identify microbial strains.

[0063] Accordingly, one aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate to obtain live cultures;(vi) optionally, identifying wells of interest with a first screen or growth and consolidating the wells of interest into at least one second multi-well plate; and (vii.A) analyzing the first multi-well plate or second multi-well plate with a first screen to identify microbial strains;(vii.B) analyzing the first multi -well plate or second multi -well plate with a second screen to identify microbial strains; and / or(vii.C) storing the live cultures of the first or second multi-well plate.

[0064] Accordingly, one aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate;(vi) identifying wells of interest with a first screen and consolidating the wells of interest into at least one second multi-well plate; and(vii.A) analyzing the second multi-well plate with a second screen to identify microbial strains; and / or1362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(vii.B) storing the second multi-well plate.

[0065] In some embodiments, the first screen comprises assaying the first multi-well plate with a first identifying assay to identify the wells of interest.

[0066] In some embodiments, the first identifying assay comprises:(A) optionally, fixing and permeabilizing a portion of the cultures from each well of interest or from a subset of wells of interest, and plating onto an array to obtain plated resuspensions, wherein the fixing and permeabilizing is done via resuspension of the cultures in ethanol;(B) providing a first set of probes, wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe;(C) contacting the first set of probes with the plated resuspensions to form a first complex;(D) imaging the array with a widefield microscope to acquire one emission spectra from the at least one first emissive readout probe; wherein the at least one first emissive readout probe emits a first color or a first set of colors, and wherein the first color or the first set of colors are assigned to a known microbrial strain

[0067] In some embodiments, the method (vii.A) analyzes the second multi-well plate with a second screen to identify microbial strains.

[0068] In some embodiments, the second screen comprises (viii) performing a second identifying assay on the second multi-well plate, wherein the second identifying assay comprises:(A) optionally, fixing and permeabilizing a portion of the cultures from each well of interest or from a subset of wells of interest, and plating onto an array to obtain plated resuspensions, wherein the fixing and permeabilizing is done via resuspension of the cultures in ethanol;(B) providing a second set of probes, wherein the second set of probes comprises at least one second encoding probe and at least one second emissive readout probe;(C) contacting the second set of probes with the wells of interest to form a second complex;1462735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(D) imaging the array with a widefield or confocal microscope to acquire one emission spectra from the at least one second emissive readout probe; wherein the at least one second emissive readout probe emits a second color or a second set of colors; and wherein the second color or second set of colors are assigned to a known microbial strain

[0069] In some embodiments, the first set of probes and second set of probes are the same.

[0070] In some embodiments, the method further comprises (ix.A) isolating cultures from wells that do not emit the second color to obtain isolate cultures.

[0071] In some embodiments, the method further comprises (ix.B) identifying cultures from wells that do not emit the second color to obtain cultures of interest.

[0072] In some embodiments, the method further comprises (ix.C) identifying cultures from wells that emit a color of interest to obtain cultures of interest.

[0073] In some embodiments, the isolate cultures or cultures of interest are diluted to obtain a diluted sample.

[0074] In some embodiments, the diluted sample is dispersed into media in a third multi-well plate for liquid growth and cultured for at least a day.

[0075] In some embodiments, the diluted sample is dispersed into media in the second multiwell plate for liquid growth and cultured for at least a day to obtain isolate cultures or cultures of interest.

[0076] In some embodiments, the method further comprises (ix.A) identifying wells of the first or second multi-well plate that emit the first or second color, or set of colors, to obtain generate a list of wells of interest.

[0077] In some embodiments, the method further comprises (ix.B) identifying wells of the first or second multi-well plate that do not emit the first or second color, or set of colors, to obtain generate a list of wells of interest.

[0078] In some embodiments, the method further comprises (ix.C) regrowing cultures from the stored live cultures of the first or second multi-well plate of step (vii.C).

[0079] In some embodiments, the method further (x) analyzes the first or second multi-well plate with a screen to identify microbial strains.

[0080] In some embodiments, the method continues the screen until one or more microbial strains of interests is confirmed to be isolated.1562735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0081] In some embodiments, the method further (x) analyzes the second or third multi-well plate with a screen to identify microbial strains, wherein the screen comprises (xi) performing a third identifying assay on the second or third multi-well plate, wherein the third identifying assay comprises:(A) resuspending the isolate cultures in ethanol and plating into an array;(B) providing a third set of probes, wherein the third set of probes comprises at least one third encoding probe and at least one third emissive readout probe;(C) contacting the third set of probes with the isolate cultures to form a third complex;(D) acquiring one or more emission spectra with a confocal microscope from the at least one third emissive readout probe;(E) determining the spectra of signal and assigning them to a species of the microbial strain;(F) repeating the aforementioned steps (xi.A)-(xi.E) for at least one different encoding probe and until the culture of interest is confirmed to be isolated.

[0082] In some embodiments, the second set of probes and third set of probes are the same.

[0083] Another aspect of the methods described herein can be used to aid in the isolation of rare or difficult to culture taxa of interest.

[0084] Accordingly, another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate;(v.A) identifying wells of interest, wherein the wells of interest are wells with growth;(vi) pooling wells of interest to obtain a pooled sample, wherein the pooling comprises removing a portion of the wells of interest;1662735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(vii) plating the pooled sample on an array;(viii) identifying a pool of interest with a first screen;(ix) isolating the pool of interest and performing steps (iii)-(v) and (vii)-(viii) on the pool of interest;(x) performing a second screen to identify a culture of interest from the pool of interest;(xi) isolating the culture of interest and performing a third screen until a microbial strain of interest is identified.

[0085] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(iv) culturing the sample for at least a day in the first multi-well plate;(v) optionally, identifying wells with growth and consolidating those wells into at least one second multi-well plate; and(vi) fixing and permeabilizing (via resuspension in ethanol) a portion of cultures from each well or a subset of the wells in the “selected” (first or, if made, second) multi-well plate,(vii) creating a pooled sample from portions of 2 or more of the above fixed and permeabilized cultures;(viii) plating the pooled sample on an array;(ix) identifying a pool of interest with a first screen;(x) plating portions or all of each of the source well cultures (generated in vi) of the pool of interest in (ix) on an array,(xi) performing a second screen to identify the source well of interest from the pool of interest;(xii) regrowing the source culture, performing a screen, and diluting / regrowing / rescreening until the microbial strain of interest is isolated.

[0086] Another aspect of the disclosure provides a method for identifying microbial strains1762735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate;(vi) pooling wells of interest to obtain a pooled sample;(vii) plating the pooled sample on an array;(viii) identifying a pool of interest with a first screen;(ix) isolating the pool of interest and performing steps (iii)-(v) and (vii)-(viii) on the pool of interest;(x) performing a second screen to identify a culture of interest from the pool of interest;(xi) isolating the culture of interest and performing a third screen until a microbial strain of interest is identified.

[0087] In some embodiments, the first, second, and third screens are performed with an identifying assay, each screen comprising:(A) providing a set of probes, wherein the set of probes comprises at least one encoding probe and at least one emissive readout probe;(B) contacting the set of probes with the pool of interest or wells of interest to form a complex;(C) imaging the array with a widefield microscope to acquire one emission spectra from the at least one emissive readout probe; wherein the at least one emissive readout probe emits a color and wherein the color is assigned to a pool of interest, well of interest, or microbial strain.

[0088] In some embodiments, the first, second, and third screens are performed with an identifying assay, each screen comprising:(A) providing a set of probes,1862735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 wherein the set of probes comprises at least one encoding probe and at least one emissive readout probe;(B) contacting the set of probes with the pool of interest or wells of interest to form a complex; and(C) visualizing the array with a widefield microscope to identify wells of interest based on which cultures have signal;(D) optionally, imaging the array with a widefield or confocal microscope to acquire one emission spectra from the at least one emissive readout probe; wherein the at least one emissive readout probe emits a color and wherein the color is assigned to a pool of interest, well of interest, or particular microbial strain.

[0089] Another aspect of the methods described herein can be used in screening taxa of interest via colony growth.

[0090] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies or biomass;(iii.A) picking live colonies and depositing the live colonies on an array; or(iii.B) picking live colonies and depositing the live colonies on a container for fixation; then transferring the fixed colonies to an array; or(iii.C) stamping an array onto the live colonies directly;(iii.C.l) optionally, fixing the colonies on the array;(iv) staining the array of any of steps (iii.A)-(iii.C);(v) performing a screen on the array of any of steps (iii.A)-(iii.C) to identify microbial strains of interest;(vi) optionally, repeating steps (iii.A)-(v); and(vii) optionally, re-culturing the microbial strain of interest from the plate in step (ii) and repeat steps (iii)-(v).

[0091] Another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies;1962735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(iii. A) picking live colonies and depositing the live colonies on an array; or(iii.B) picking live colonies and depositing the live colonies on a container for fixation; then transferring the fixed colonies to an array; or(iii.C) stamping an array onto the live colonies directly;(iv) staining the array of any of steps (iii. A)-(iii.C);(v) performing a screen on the array of any of steps (iii.A)-(iii.C) to identify microbial strains of interest;(vi) optionally, repeating steps (iii.A)-(v); and(vii) optionally, re-culturing the microbial strain of interest and repeat steps (ii)-(v).

[0092] In some embodiments, steps (iii.A)-(v) are repeated.

[0093] In some embodiments, the microbial strain of interest is re-cultured and steps (ii)-(v) are repeated.

[0094] In some embodiments, to reduce disturbance to the colonies and increase biomass available for further isolation a replica plating strategy is be employed. In some embodiments, one replica plate is stored live and the other replica plate is used for screening with the methods described herein.

[0095] Accordingly, another aspect of the disclosure provides a method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies;(iii) stamping a first array onto the live colonies directly;(iv) stamping a second array onto the first array;(v) generating at least one replica plate by stamping the first array onto a new plate;(vi) fixing and staining the second array;(vii) performing a screen on the second array to identify microbial strains of interest;(viii) optionally, repeating steps (iii)-(v); and(ix) optionally, re-culturing the microbial strain of interest and repeat steps (ii)-(v).

[0096] In some embodiments, at least two replica plates are generated. In some embodiments, two replica plates are generated. In some embodiments, a first replica plate is generated, wherein the first replica plate is stored live for isolation of microbial strains. In some embodiments, a second replica plate is generated, wherein the second replica plate is used for screening with the2062735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 methods described herein.

[0097] In some embodiments, once the microbial strains of interest have been identified in the second array, the location of said microbial strains of interest can be used to guide colony picking from the live replica plate.

[0098] In some embodiments of this aspect, step (iii) is replaced by (iii.A), wherein in (iii.A) live colonies are picked and the live colonies are deposited on a first array. In some embodiments of this aspect, step (iii) is replaced by (iii.B), wherein in (iii.B) live colonies are picked and the live colonies are deposited on a container for fixation; then the fixed colonies are transferred to a first array.

[0099] In some embodiments of this aspect, step (iv) is replaced by (iv.A), wherein in (iv.A) live colonies are picked and the live colonies are deposited on a first array. In some embodiments of this aspect, step (iv) is replaced by (iv.B), wherein in (iv.B) live colonies are picked and the live colonies are deposited on a container for fixation; then the fixed colonies are transferred to a first array.

[0100] In some embodiments, steps (iii)-(v) are repeated.

[0101] In some embodiments, the microbial strain of interest is re-cultured and steps (ii)-(v) are repeated.

[0102] In some embodiments, when the live colonies are deposited for fixation, the fixative is selected from a formaldehyde fixative or an ethanol spray fixative. In some embodiments, when the live colonies are deposited for fixation, the fixative is a formaldehyde fixative. In some embodiments, when the live colonies are deposited for fixation, the fixative is an ethanol spray fixative.

[0103] In some embodiments, the method further comprises analyzing if the microbial strain is part of a mixed-species culture. If the microbial strain is part of a mixed-species culture, the proportion of the total population of the microbial strain of interest is analyzed.

[0104] In some embodiments, a “mixed-species culture” includes cultures from one microbial group. In some embodiments, a “mixed-species culture” includes a mixture of organisms, such as fungus and bacteria.

[0105] In some embodiments, if the microbial strain is part of a mixed-species culture, the culture is further cultured on agar media for isolation of clonal population.

[0106] In some embodiments, prior to performing the first screen a reference panel for2162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 comparison is prepared.

[0107] In some embodiments, the reference panel is prepared by:(i) extracting DNA or RNA from an aliquot of the sample to obtain a sequencing panel;(ii) performing long read sequencing on the sequencing panel to obtain RNA sequencing data; and(iii) designing encoding probes based on the obtained RNA sequencing data, wherein each encoding probe comprises a targeting sequence; and(iv) selecting the encoding probes to reflect the reference panel.Analysis

[0108] In some embodiments, the encoding probes are imaged by measuring emission spectra emitted from the readout probes. In some embodiments, the methods comprise acquiring one or more emission spectra with a confocal microscope from the at least one emissive readout probe. In some embodiments, the methods determine the spectra of signal and assigning them to a species of the microbial strain. In some embodiments, the methods comprise decoding the spectra into a single barcode representative of a specific taxa or microbial strain through means of signal deconvolution, error correction, comparison to reference standards.

[0109] In some embodiments, in addition to or instead of measuring spectra, the data is processed and features of the microbes (including but not limited to spectra) are used to characterize the samples. For example, pan-microbial stain can be used. In some embodiments, pan-microbial stain allows for segmentation of all of the microbes in an image; then, morphological features can be used to aid classification of the microbes, and abundance analysis can be conducted to determine what percent of mixed sample is represented by the taxon of interest.

[0110] In some embodiments, the pan-microbial stain is 4’,6-diamidino-2-phenylindole (DAPI)

[0111] In some embodiments, in addition to or instead of measuring spectra, a viability dye is used to determine whether the culture or colony will be likely to regrow in further isolation efforts.

[0112] A “viability dye” stains any cell that is permeable and dead, thus detecting the signal assesses cell viability. When using fixable dyes, there are two approaches for analyzing viability.2262735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924In some embodiments, the viability of cells is assessed via a relative signal analysis or a barcode classification.

[0113] Relative Signal Analysis: Assesses the amount of viability dye signal relative to a nuclear stain, such as DAPI. The viability dye signal — measured either by peak intensity or total integrated intensity — can be used to determine the viability status of the cells.

[0114] In some embodiments, the relative signal analysis measures the ratio between the amount of viability dye signal relative to the amount of nuclear dye signal.

[0115] Barcode Classification: Treat the viability dye as a unique “bit” in a barcode system, where dead cells register this bit as “1” and live cells as “0.” The first digit of the cell barcode is reserved for viability (e.g., X101, where “101” represents taxonomic identification, such as E. coli). In this system, “0101” represents a live E. coli, while “1101” represents a dead E. coli.

[0116] In some embodiments, the barcode classification assigns a bit to the viability dye, wherein the bit “1” is assigned to dead cells and bit “0” is assigned to live cells.

[0117] In some embodiments, the methods described herein further comprise incorporating a viable dye. In some embodiments, the methods described herein further comprise incorporating controls into the sample prior to imaging, wherein the controls comprise community -based controls or strain-specific controls.

[0118] The detection of dead cells in the present methods is based on cell wall permeability, which varies across different taxa, leading to differential absorption of viability dyes (also with DAPI). To account for this variability, controls can be used with each experimental condition to distinguish live from dead cells. Two strategies for incorporating controls can be used:

[0119] Community -Based Controls: For each microbial community, a “live” and “dead” control is created by dividing the initial sample into three parts at the beginning of the method described herein. One part serves as the test sample. The other two parts are immediately fixed and washed — one without exposure to viability dye (serving as a “live” control), and the other stained with viability dye (representing a “dead” control). Since all cells in these controls are permeabilized during fixation, the viability dye stains only the dead cells. All three samples undergo image processing, and a viability measurement is performed for each taxon to assess accuracy.

[0120] In some embodiments, the community-based control comprises a preparation of a “live control” and a “dead control,” wherein the “live control” comprises a portion of the sample that is2362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 fixed and washed and has not been exposed to a viability dye; and wherein the “dead control” comprises a portion of the sample that is fixed and washed and has been exposed to a viability dye.

[0121] Strain-Specific Controls: In here, each pure stock can be used to generate both live and dead controls in the presence of viability dye. This information is then used to build a reference database, against which experimental samples can be compared to ensure accurate viability assessments.

[0122] In some embodiments, the Strain-Specific Control comprises a preparation of “live pure culture” and “dead pure culture” of a known, individual strain, wherein the “live pure culture” comprises a portion of a culture of a known, individual strain that is fixed and washed and has not been exposed to a viability dye; and wherein the “dead pure culture” comprises a portion of a culture of a known, individual strain that is fixed and washed and has been exposed to a viability dye.

[0123] In some embodiments, a detectable signal derives from the viability dye and is indicative of the presence of dead cells. In some embodiments, the viability dye is imaged by measuring emission spectra emitted from the viability dye. In some embodiments, the detectable signal is a fluorescent signal at a specific wavelength (nm).

[0124] In some embodiments, the detectable signal derives from the viability dye and is indicative of the presence of live cells. In some embodiments, the viability dye is imaged by measuring emission spectra emitted from the viability dye. In some embodiments, the first detectable signal is a fluorescent signal at a specific wavelength (nm).

[0125] In some embodiments, the viability dye comprises LIVE / DEAD® Fixable Blue stain, LIVE / DEAD® Fixable Violet stain, LIVE / DEAD® Fixable Lime stain, LIVE / DEAD® Fixable Aqua stain, LIVE / DEAD® Fixable Yellow stain, LIVE / DEAD® Fixable Green stain, LIVE / DEAD® Fixable Olive stain, LIVE / DEAD® Fixable Orange stain, LIVE / DEAD® Fixable Red stain, LIVE / DEAD® Fixable Far Red stain, LIVE / DEAD® Fixable Scarlet stain, LIVE / DEAD® Fixable Near-IR (775) stain, LIVE / DEAD® Fixable Near-IR (780) stain, LIVE / DEAD® Fixable Near IR (876) stain, or 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC).

[0126] In some embodiments, the viability dye is a LIVE / DEAD®™ Fixable Blue stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Violet stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Lime stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Aqua stain. In some embodiments, the viability dye is a2462735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924LIVE / DEAD® Fixable Yellow stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Green stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Olive stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Orange stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Red stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Far Red stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Scarlet stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Near-IR (775) stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Near-IR (780) stain. In some embodiments, the viability dye is a LIVE / DEAD® Fixable Near IR (876) stain. In some embodiments, the viability dye is 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC). LIVE / DEAD® Fixable stains are commercially available from ThermoFisher Scientific.Sample

[0127] In some embodiments, the sample comprises at least one cell, a cell suspension, a tissue biopsy, a tissue specimen, bone biopsies, organoids, three-dimensional hydrogel scaffolds, transwell systems, or plant biopsies.

[0128] In some embodiments, the sample is a cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a unicellular organism including protozoa, chromista, algae, or fungi. In some embodiments, the eukaryotic cell is part of a multicellular organism from chromista, plantae, fungi, or animalia. In some embodiments, the sample is a tissue composed of cells. In some embodiments, the cell contains foreign DNA / RNA from viruses, plasmids, and bacteria. In some embodiments, the cell has been genetically modified or synthetically engineered. In some embodiments, the cell has been genetically modified.

[0129] In some embodiments, the sample can include a plurality of cells. In some embodiments, each cell in the plurality of cells can include a specific targeting sequence, which may or may not be the same from the other targeting sequences.

[0130] In some embodiments, the sample is a whole organism.

[0131] In some embodiments, the sample comprises a plurality of cells. In some embodiments, the sample comprises a plurality of cells that contain the pathogen.

[0132] In some embodiments, the sample is obtained from a patient diagnosed with, or suspected to be suffering from an infection, disease, or disorder. In some embodiments, the patient2562735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 has been diagnosed with, or is suspected to be suffering from a bacterial, viral, fungal, or parasitic infection. In some embodiments, the infection includes, but is not limited to, Acute Flaccid Myelitis, Anaplasmosis, Anthrax, Babesiosis, Botulism, Brucellosis, Campylobacteriosis, Carbapenem-resistant Infection (CRE / CRPA), Chancroid, Chickenpox, Chikungunya Virus Infection (Chikungunya), Chlamydia, Ciguatera (Harmful Algae Blooms (HABs)), Clostridium Difficile Infection, Clostridium Perfringens (Epsilon Toxin), Coccidioidomycosis fungal infection (Valley fever), COVID-19 (Coronavirus Disease 2019), Creutzfeldt-Jacob Disease, transmissible spongiform encephalopathy (CJD), Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue, 1,2, 3, 4 (Dengue Fever), Diphtheria, E. coli infection, Shiga toxin-producing (STEC), Eastern Equine Encephalitis (EEE), Ebola Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Enterovirus Infection , D68 (EV-D68), Enterovirus Infection, Non-Polio (NonPolio Enterovirus), Giardiasis (Giardia), Glanders, Gonococcal Infection (Gonorrhea), Granuloma inguinale, Haemophilus Influenza disease, Type B (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis (A, B, C, D, and / or E), Herpes Herpes Zoster, zoster VZV (Shingles), Histoplasmosis infection (Histoplasmosis), Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Lead Poisoning, Legionellosis (Legionnaires Disease), Leishmaniasis, Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma venereum infection (LGV), Malaria, Measles, Melioidosis, Meningitis, Viral (Meningitis, viral), Meningococcal Disease , Bacterial (Meningitis, bacterial), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Mononucleosis, Multisystem Inflammatory Syndrome in Children (MIS-C), Mumps, Norovirus, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pediculosis (Lice, Head and Body Lice), Pelvic Inflammatory Disease (PID), Pertussis (Whooping Cough), Plague; Bubonic, Septicemic, Pneumonic (Plague), Pneumococcal Disease (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis (Parrot Fever), Phthiriasis (Crabs; Pubic Lice Infestation), Pustular Rash diseases (Small pox, monkeypox, cowpox), Q-Fever, Rabies, Ricin Poisoning, Rickettsiosis (Rocky Mountain Spotted Fever), Rubella, Salmonellosis gastroenteritis (Salmonella), Scabies Infestation (Scabies), Scombroid, Septic Shock (Sepsis), Severe Acute Respiratory Syndrome (SARS), Shigellosis gastroenteritis (Shigella), Smallpox, Staphylococcal Infection , Methicillin-resistant (MRSA), Staphylococcal Food Poisoning, Enterotoxin - B Poisoning (Staph Food Poisoning), Staphylococcal Infection, Vancomycin2662735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924Intermediate (VISA), Staphylococcal Infection, Vancomycin Resistant (VRSA), Streptococcal Disease , Group A (invasive) (Strep A (invasive)), Streptococcal Disease, Group B (Strep-B), Streptococcal Toxic-Shock Syndrome, STSS, Toxic Shock (STSS, TSS), Syphilis , primary, secondary, early latent, late latent, congenital, Tetanus, Toxoplasmosis, Trichomoniasis (Trichomonas infection), Trichinosis Infection (Trichinosis), Tuberculosis (Latent) (LTBI), Tuberculosis (TB), Tularemia (Rabbit fever), Typhus, Typhoid Fever, Group D, Vaginosis , bacterial (Yeast Infection), Vaping-Associated Lung Injury (e-Cigarette Associated Lung Injury), Varicella (Chickenpox), Vibrio cholerae (Cholera), Vibriosis (Vibrio), Viral Hemorrhagic Fever (Ebola, Lassa, Marburg), West Nile Virus, Yellow Fever, Yersenia (Yersinia), or Zika Virus Infection (Zika).

[0133] In some embodiments, when the sample is obtained from a patient, the patient has been diagnosed with, or is suspected to be suffering from an infection caused by a bacterium selected from the group consisting of: Acinetobacter, Actinomyces, Aerococcus, Bacteroides, Bartonella, Brucella, Bordetella, Burkholderia, Campylobacter, Chlamydia, Citrobacter, Clostridium, Corynebacterium, Edwardsiella, Elizabethkingia, Enterobacter, Enterococcus, Escherichia, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Morganella, Mycobacterium, Mycoplasma, Neisseria, Pantoea, Prevotella, Proteus, Providencia, Pseudomonas, Raoultella, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Ureaplasma, and Vibrio.

[0134] In some embodiments, when the sample is obtained from a patient, the patient has been diagnosed with, or is suspected to be suffering from an infection caused by a virus selected from the group consisting of: bacteriophage, RNA bacteriophage (e.g., MS2, AP205, PP7 and Q(3), Infectious Haematopoietic Necrosis Virus, Parvovirus, Herpes Simplex Virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Measles virus, Mumps virus, Rubella virus, HIV, Influenza virus, Rhinovirus, Rotavirus A, Rotavirus B, Rotavirus C, Respiratory Syncytial Virus (RSV), Varicella zoster, and Poliovirus, Norovirus, Zika virus, Dengue Virus, Rabies Virus, Newcastle Disease Virus, and White Spot Syndrome Virus.

[0135] In some embodiments, when the sample is obtained from a patient, the patient has been diagnosed with, or is suspected to be suffering from an infection caused by a parasite selected from the group consisting of: Plasmodium, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, helminthic parasites: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma2762735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. and Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp.

[0136] In some embodiments, the sample is obtained from a patient diagnosed with, or suspected to be suffering from cancer. In some embodiments, the patient is being treated for cancer. In some embodiments, the patient has been treated for cancer. In some embodiments, the patient has been treated with one or more therapeutic agents, such as, but not limited to an inhibitory immune checkpoint blocker or inhibitor, a stimulatory immune checkpoint stimulator, agonist or activator, a chemotherapeutic agent, an anti-cancer agent, a radiotherapeutic agent, an anti- neoplastic agent, an anti-proliferation agent, an anti- angiogenic agent, an anti-inflammatory agent, an immunotherapeutic agent, a therapeutic antigen-binding molecule (mono- and multispecific antibodies and fragments thereof in any format (e.g., including without limitation DARTs®, Duobodies®, BiTEs®, BiKEs, TriKEs, XmAbs®, TandAbs®, scFvs, Fabs, Fab derivatives), bispecific antibodies, non-immunoglobulin antibody mimetics (e.g., including without limitation adnectins, affibody molecules, affilins, affimers, affitins, alphabodies, anticalins, peptide aptamers, armadillo repeat proteins (ARMs), atrimers, avimers, designed ankyrin repeat proteins (DARPins®), fynomers, knottins, Kunitz domain peptides, monobodies, and nanoCLAMPs), antibody-drug conjugates (ADC), antibody -peptide conjugate), an oncolytic virus, a gene modifier or editor, a cell comprising a chimeric antigen receptor (CAR), e.g., including a T cell immunotherapeutic agent, an NK-cell immunotherapeutic agent, or a macrophage immunotherapeutic agent, a cell comprising an engineered T-cell receptor (TCR-T), or any combination thereof.

[0137] In some embodiments, the sample is a stool sample from a human donor.

[0138] In some embodiments, the microbial strain is a strain derived from a microbe selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism (e.g., wherein specific genes are added, deleted, mutated, driven to high expressions, and / or suppressed to low expression levels). In some embodiments, the microbial strain is a bacterial strain. In some embodiments, the bacterial strain is a probiotic strain.

[0139] In some embodiments, the microbial strain is a genetically engineered strain. In a “genetically engineered” strain, specific genes are added, deleted, mutated, driven to high2862735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 expressions, and / or suppressed to low expression levels. The alterations are known to a person of ordinary skill in the art.

[0140] In some embodiments, the microbial strain is a genetically engineered bacterial strain. In some embodiments, the genetically engineered strain is a genetically engineered bacterial strain. In some embodiments, the microbial strain is a genetically engineered microbial strain. In some embodiments, the genetically engineered strain is a genetically engineered microbial strain. In some embodiments, the genetically engineered strain is green fluorescence protein (GFP) E. coli.

[0141] In some embodiments, the sample is diluted by at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In some embodiments, the sample is diluted by at least 5-fold. In some embodiments, the sample is diluted by at least 6-fold. In some embodiments, the sample is diluted by at least 7-fold. In some embodiments, the sample is diluted by at least 8-fold. In some embodiments, the sample is diluted by at least 9-fold. In some embodiments, the sample is diluted by at least 10-fold.

[0142] In some embodiments, the sample is diluted 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In some embodiments, the sample is diluted 5-fold. In some embodiments, the sample is diluted 6-fold. In some embodiments, the sample is diluted 7-fold. In some embodiments, the sample is diluted 8-fold. In some embodiments, the sample is diluted 9-fold. In some embodiments, the sample is diluted 10-fold.

[0143] In some embodiments, the sample is diluted by at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least a 1000-fold, at least a 10,000-fold, or at least a 100,000-fold.

[0144] In some embodiments, the sample is diluted from about 10,000-fold to about 100,000- fold. In some embodiments, the sample is diluted about 10,000-fold, about 20,000-fold, about 30,000-fold, about 40,000-fold, about 50,000-fold, about 60,000-fold, about 70,000-fold, about 80,000-fold, about 90,000-fold, or about 100,000-fold.Encoding Probe Hybridization

[0145] The methods described herein include providing a first, second and / or third set of probes. In some embodiments, the set of probes comprises at least one encoding probe and at least one emissive readout probe. In some embodiments, the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe. In some embodiments, the2962735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 second set of probes comprises at least one second encoding probe and at least one second emissive readout probe. In some embodiments, the third set of probes comprises at least one third encoding probe and at least one third emissive readout probe.

[0146] Encoding probes, for example, are designed to target bacterial ribosomal RNA (rRNA) and messenger RNA (mRNA) targets.

[0147] For example, rRNA-probes can contain (5’ to 3’): a. Primer sequences to enrich probe pool. b. A readout-complementary sequence. c. rRNA target complementary sequence. d. A readout-complementary sequence (can be same or different than b). e. Primer sequences to enrich probe pool.

[0148] mRNA-probes contain (5’ to 3’): a. Primer sequences to enrich probe pool. b. An initiator sequence. c. mRNA target complementary sequence. d. An initiator sequence (can be same or different than b). e. Primer sequences to enrich probe pool.

[0149] Primer Sequences

[0150] In some embodiments, the primer sequence can include about 10 to about 30, about 15 to about 25, about 18 to about 23, about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long.

[0151] Targeting Sequence

[0152] In some embodiments, each of the encoding probes comprise a targeting sequence.

[0153] In some embodiments, the targeting sequence targets at least one of messenger RNA (mRNA), micro RNA (miRNA), long non coding RNA (IncRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), transfer RNA (tRNA), Crispr RNA (crRNA), trans-activating cirspr RNA (tracrRNA), mitochondria RNA, Intronic RNA, viral mRNA, viral genomic RNA, environmental RNA, double-stranded RNA (dsRNA), small nuclear RNA (snRNA), small nucleolar (snoRNA), piwi-interacting RNA (piRNA), genomic DNA, synthetic DNA, DNA, plasmid DNA, a plasmid, viral DNA, retroviral DNA, environmental DNA, extracellular DNA, a protein, a small molecule, or an antigenic target. In some embodiments, the target is mRNA. In some embodiments, the target is rRNA. In some embodiments, the target is mRNA and rRNA.

[0154] In some embodiments, the targeting sequence targets messenger RNA (mRNA). In some embodiments, the targeting sequence targets micro RNA (miRNA). In some embodiments,3062735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 the targeting sequence targets long non-coding RNA (IncRNA). In some embodiments, the targeting sequence targets ribosomal RNA (rRNA). In some embodiments, the targeting sequence targets small interfering RNA (siRNA). In some embodiments, the targeting sequence targets transfer RNA (tRNA). In some embodiments, the targeting sequence targets Crispr RNA (crRNA). In some embodiments, the targeting sequence targets trans-activating Crispr RNA (tracrRNA). In some embodiments, the targeting sequence targets mitochondrial RNA. In some embodiments, the targeting sequence targets intronic RNA. In some embodiments, the targeting sequence targets viral mRNA. In some embodiments, the targeting sequence targets viral genomic RNA. In some embodiments, the targeting sequence targets environmental RNA. In some embodiments, the targeting sequence targets double-stranded RNA (dsRNA). In some embodiments, the targeting sequence targets small nuclear RNA (snRNA). In some embodiments, the targeting sequence targets small nucleolar RNA (snoRNA). In some embodiments, the targeting sequence targets piwi-interacting RNA (piRNA). In some embodiments, the targeting sequence targets genomic DNA. In some embodiments, the targeting sequence targets synthetic DNA. In some embodiments, the targeting sequence targets DNA. In some embodiments, the targeting sequence targets plasmid DNA. In some embodiments, the targeting sequence targets a plasmid. In some embodiments, the targeting sequence targets viral DNA. In some embodiments, the targeting sequence targets retroviral DNA. In some embodiments, the targeting sequence targets environmental DNA. In some embodiments, the targeting sequence targets extracellular DNA. In some embodiments, the targeting sequence targets a protein. In some embodiments, the targeting sequence targets a small molecule. In some embodiments, the targeting sequence targets an antigenic target.

[0155] In some embodiments, the targeting sequence targets mRNA and / or rRNA. In some embodiments, the targeting sequence targets mRNA. In some embodiments, the targeting sequence targets rRNA. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23 S rRNA sequence in the microbial strain. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23S rRNA sequence in a bacterial strain. In some embodiments, the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23S rRNA sequence in the pathogen.

[0156] In some embodiments, the targeting sequence targets a 18S rRNA sequence, a 5.8S rRNA sequence, and / or a 28S rRNA sequence in the in the microbial strain, wherein the microbial strain is a eukaryotic microbial strain. In some embodiments, the targeting sequence targets a 18S3162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 rRNA sequence. Tn some embodiments, the targeting sequence targets a 5.8S rRNA sequence. In some embodiments, the targeting sequence targets a 28S rRNA sequence.

[0157] In some embodiments, the targeting sequence of the encoding probe is substantially complementary to a specific target sequence. By "substantially complementary" it is meant that the nucleic acid fragment is capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase.

[0158] In some embodiments, to hybridize the encoding probes to the complex, encoding buffer is added to the sample. In some embodiments, the encoding buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, a blocking agent, or combinations thereof. In some embodiments, the encoding buffer includes more than one type of agent, for example, the encoding buffer can include two or more polyanionic polymers and / or two or more blocking agents.Readout Probe Hybridization

[0159] After the encoding hybridization step is complete, the methods described herein acquire one or more emission spectra from the at least one first, second, or third emissive readout probe.

[0160] In some embodiments, each emissive readout probe can include a label and a complementary sequence to the readout sequence of the encoding probe. In some embodiments, this step may be referred to as the “readout probe hybridization” step. In here, the emissive readout probes hybridize to their complementary sequences present in the first or second complex.

[0161] Emissive readouts probes are 10-50 nucleotide-long oligonucleotides bound with one of eleven fluorescent dyes at the 5’ - and / or 3’ - end.

[0162] Readout probes can be designed as follows: a. Are coupled to 1, 2, or more fluorescent dyes. b. Are orthogonal to all biological sequences. c. Are orthogonal to each other / each other’s complementary sequences.

[0163] In some embodiments, the emissive readout sequence is about 10 to about 50, about 15 to about 50, about 15 to about 45, about 15 to about 35, about 15 to about 30, about 18 to about 24, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.

[0164] In some embodiments, the emissive readout probe can include a label on the 5’ or 3’ end. In some embodiments, the emissive readout probe can include a label on the 5’ end and a label on the 3’ end. In some embodiments, the labels are the same. In some embodiments, the3262735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 labels are different.

[0165] In some embodiments, the label is a fluorescent entity (fluorophore) or phosphorescent entity. In some embodiments, the label is a cyanine dye (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dye, Atto dye, photoswitchable dye, photoactivatable dye, fluorescent dye, metal nanoparticle, semiconductor nanoparticle or "quantum dots", fluorescent protein such as GFP (Green Fluorescent Protein), or photoactivatable fluorescent protein, such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PAmCherry, PAtagRFP, mMaple, mMaple2, and mMaple3.

[0166] In some embodiments, the label is Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647-R-phycoerythrin, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-allophycocyanin, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor Plus 405, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 594, Alexa Fluor Plus 647, Alexa Fluor Plus 680, Alexa Fluor Plus 750, Alexa Fluor Plus 800, Pacific Blue, Pacific Green, Rhodamine Red X, DyLight 485-LS, DyLight-510-LS, DyLight 515-LS, DyLight 521-LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Fluorescein FITC, R-phycoerythrin (PE), Tamara, Cy3.5 581 , Rox, Red 613, Texas Red, Cy5, Cy5.5, Cy7, Allophycocyanin, ATTO 430LS, ATTO 490LS, ATTO 390, ATTO 425, Cyan 500 NHS-Ester, ATTO 465, ATTO 488, ATTO 495, ATTO Rhol lO, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740.

[0167] In some embodiments, the label is Alexa Fluor 350. In some embodiments, the label is Alexa Fluor 405. In some embodiments, the label is Alexa Fluor 430. In some embodiments, the label is Alexa Fluor 488. In some embodiments, the label is Alexa Fluor 514. In some embodiments, the label is Alexa Fluor 532. In some embodiments, the label is Alexa Fluor 546. In some embodiments, the label is Alexa Fluor 555. In some embodiments, the label is Alexa Fluor 561. In some embodiments, the label is Alexa Fluor 568. In some embodiments, the label is Alexa Fluor 594. In some embodiments, the label is Alexa Fluor 610. In some embodiments, the label is3362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924Alexa Fluor 633. In some embodiments, the label is Alexa Fluor 635. In some embodiments, the label is Alexa Fluor 647. In some embodiments, the label is Alexa Fluor 647-R-phycoerythrin. In some embodiments, the label is Alexa Fluor 660. In some embodiments, the label is Alexa Fluor 680. In some embodiments, the label is Alexa Fluor 680-allophycocyanin. In some embodiments, the label is Alexa Fluor 700. In some embodiments, the label is Alexa Fluor 750. In some embodiments, the label is Alexa Fluor 790. In some embodiments, the label is Alexa Fluor Plus 405. In some embodiments, the label is Alexa Fluor Plus 488. In some embodiments, the label is Alexa Fluor Plus 555. In some embodiments, the label is Alexa Fluor Plus 594. In some embodiments, the label is Alexa Fluor Plus 647. In some embodiments, the label is Alexa Fluor Plus 680. In some embodiments, the label is Alexa Fluor Plus 750. In some embodiments, the label is Alexa Fluor Plus 800. In some embodiments, the label is Pacific Blue. In some embodiments, the label is Pacific Green. In some embodiments, the label is Rhodamine Red X. In some embodiments, the label is DyLight 485-LS. In some embodiments, the label is DyLight-510-LS. In some embodiments, the label is DyLight 515-LS. In some embodiments, the label is DyLight 521-LS. In some embodiments, the label is Hydroxy coumarin. In some embodiments, the label is methoxycoumarin. In some embodiments, the label is Cy2. In some embodiments, the label is FAM. In some embodiments, the label is Fluorescein FITC. In some embodiments, the label is R- phycoerythrin (PE). In some embodiments, the label is Tamara. In some embodiments, the label is Cy3.5 581. In some embodiments, the label is Rox. In some embodiments, the label is Red 613. In some embodiments, the label is Texas Red. In some embodiments, the label is Cy5. In some embodiments, the label is Cy5.5. In some embodiments, the label is Cy7. In some embodiments, the label is Allophycocyanin. In some embodiments, the label is ATTO 430LS. In some embodiments, the label is ATTO 490LS. In some embodiments, the label is ATTO 390. In some embodiments, the label is ATTO 425. In some embodiments, the label is Cyan 500 NHS-Ester. In some embodiments, the label is ATTO 465. In some embodiments, the label is ATTO 488. In some embodiments, the label is ATTO 495. In some embodiments, the label is ATTO Rhol 10. In some embodiments, the label is ATTO 514. In some embodiments, the label is ATTO 520. In some embodiments, the label is ATTO 532. In some embodiments, the label is ATTO Rho6G. In some embodiments, the label is ATTO 542. In some embodiments, the label is ATTO 550. In some embodiments, the label is ATTO 565. In some embodiments, the label is ATTO Rho3B. In some embodiments, the label is ATTO Rhol 1. In some embodiments, the label is ATTO Rhol2. In some3462735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 embodiments, the label is ATTO Thiol 2. Tn some embodiments, the label is ATTO RholOl . In some embodiments, the label is ATTO 590. In some embodiments, the label is ATTO 594. In some embodiments, the label is ATTO Rhol3. In some embodiments, the label is ATTO 610. In some embodiments, the label is ATTO 620. In some embodiments, the label is ATTO Rhol4. In some embodiments, the label is ATTO 633. In some embodiments, the label is ATTO 643. In some embodiments, the label is ATTO 647. In some embodiments, the label is ATTO 647N. In some embodiments, the label is ATTO 655. In some embodiments, the label is ATTO Oxal2. In some embodiments, the label is ATTO 665. In some embodiments, the label is ATTO 680. In some embodiments, the label is ATTO 700. In some embodiments, the label is ATTO 725. In some embodiments, the label is ATTO 740.

[0168] In some embodiments, to hybridize the readout probes to the first, second, or third complex, readout buffer is added to the sample. In some embodiments, the readout buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, a blocking agent, or combinations thereof. In some embodiments, the readout buffer includes more than one type of agent, for example, the readout buffer can include two or more poly anionic polymers and / or two or more blocking agents.

[0169] In some embodiments, after each reaction and before proceeding to the next one, the samples or probes are washed with a “wash buffer.”

[0170] In some embodiments, the wash buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, acids, a pH stabilizer, a chelating agent, or combinations thereof. In some embodiments, the wash buffer can include more than one type of agent, for example, the wash buffer can include two or more detergents. In some embodiments, the wash buffer can include a denaturing / deionizing agent, a salt buffer, a detergent, a polyanionic polymer, and an acid. In some embodiments, the wash buffer can include a salt buffer and a detergent. In some embodiments, the wash buffer can include a salt buffer, a pH stabilizer, and a chelating agent.

[0171] In some embodiments, the emissive readout probes are imaged using widefield epifluorescence microscopy, widefield microscopy, or whole-slide scanner.

[0172] In some embodiments, the emissive readout probe is imaged using point scanning confocal microscopy, spinning disk confocal microscopy, lattice lightsheet microscopy, or light field microscopy.3562735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0173] In some embodiments, specimens imaged with the above modalities can be imaged at different spatial resolution using different microscope objectives including lOx, 20x, 32x, 40x, 63x, or lOOx, which can be compatible with air, oil, or water immersion medium.

[0174] In some embodiments, the detection strategy used is channel, spectral, channel and fluorescence lifetime, or spectral and fluorescence lifetime.

[0175] In some embodiments, the array is a microscope slide, at least one chamber, at least one microfluidic device, at least one well, at least one plate, at least one filter membrane, at least one cover slip, or at least one adhesive tape. In some embodiments, the array is a microscope slide. In some embodiments, the array is at least one chamber. In some embodiments, the array is at least one microfluidic device. In some embodiments, the array is at least one well. In some embodiments, the array is at least one plate. In some embodiments, the array is at least one filter membrane. In some embodiments, the array is at least one adhesive tape. In some embodiments, the array is at least one piece of adhesive tape.

[0176] In some embodiments, the array is pre-treated with a coating to enhance adhesion of microbes (i.e. poly-d-lysine).

[0177] In some embodiments, the sample is cultured until growth is detected in at least 51%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 51% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 55% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 60% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 70% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 75% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 80% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 85% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 90% of the wells of the multi-well plate. In some embodiments, the sample is cultured until growth is detected in at least 95% of the wells of the multi-well plate.

[0178] In some embodiments, the sample is cultured for at least one day. In some3662735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 embodiments, the sample is cultured for one day. In some embodiments, the sample is cultured for two days.

[0179] In some embodiments, the sample is cultured as growth is detected in groups of wells from the first multi-well plate. In some embodiments, the sample is cultured until growth is detected in groups of wells from the first multi-well plate.

[0180] In some embodiments, the first multi-well plate is a 6-well plate, 8-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, or a 1536-well plate.

[0181] In some embodiments, the first multi-well plate is a 6-well plate. In some embodiments, the first multi-well plate is an 8-well plate. In some embodiments, the first multi-well plate is a 12- well plate. In some embodiments, the first multi-well plate is a 24-well plate. In some embodiments, the first multi-well plate is a 48-well plate. In some embodiments, the first multiwell plate is a 96-well plate. In some embodiments, the first multi -well plate is a 384-well plate. In some embodiments, the first multi-well plate is a 1536-well plate.

[0182] In some embodiments, the second plate is a single-well plate or a multi-well plate. In some embodiments, the second multi -well plate is a 6-well plate, 8-well plate, 12-well plate, 24- well plate, 48-well plate, 96-well plate, 384-well plate, or a 1536-well plate.

[0183] In some embodiments, the second multi-well plate is a 6-well plate. In some embodiments, the second multi-well plate is an 8-well plate. In some embodiments, the second multi-well plate is a 12-well plate. In some embodiments, the second multi-well plate is a 24-well plate. In some embodiments, the second multi-well plate is a 48-well plate. In some embodiments, the second multi-well plate is a 96-well plate. In some embodiments, the second multi-well plate is a 384-well plate. In some embodiments, the second multi-well plate is a 1536-well plate.

[0184] In some embodiments, the methods described herein further comprise isolating the identified microbial species and incorporating into a strain bank.

[0185] In some embodiments, the methods described herein further comprise performing whole genome sequencing on the isolated microbial species to obtain strain-level genetic information.

[0186] In some embodiments, synthetic strains are prepared based on the genetic information obtained by the whole genome sequencing.Barcoded Probes3762735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0187] The encoding probes used in the methods described herein use barcoded probes. The barcoded probes represent a probe / sequence that is specific to a target sequence in the sample / complex with a unique code.

[0188] In some embodiments, the barcoded probes include the encoding probes and readout sequences described herein.

[0189] In some embodiments, each sample or target in the sample to be identified is assigned a unique n-bit binary code selected from a plurality of unique n-bit binary codes, where n is an integer equal to or greater than 1. In some embodiments, each encoding probe does not have a unique binary code, instead groups of or all of the encoding probes have the same color.

[0190] A "binary code" refers to a representation of target sequence in a sample using a string made up of a plurality of "0" and "1" from the binary number system. The binary code is made up of a pattern of n binary digits (n-bits), where n is an integer representing the number of labels used. The bigger the number n, the greater number of targets can be represented using the binary code. For example, a binary code of eight bits (an 8-bit binary code, using 8 different labels) can represent up to 255 (28- 1) possible targets. (One is subtracted from the total possible number of codes because no target sequence is assigned a code of all zeros "00000000." A code of all zeros would mean no decoding sequence, and thus no label, is attached. In other words, there are no nonlabeled target sequences.) Similarly, a binary code of ten bits (a 10-bit binary code) can represent up to 1023 (210- 1) possible target sequences. In some embodiments a binary code may be translated into and represented by a decimal number. For example, the 10-bit binary code "0001100001" can also be represented as the decimal number "97."

[0191] Each digit in a unique binary code represents whether a readout probe and the fluorophore corresponding to that readout probe are present for the selected strain or taxa. In some embodiments, each digit in the binary code corresponds to a Readout probe (from Readout probe 1 (Rl) through Readout probe n (Rn) in an n-bit coding scheme). In a specific embodiment, the n is 10 and the digits of an n-bit code correspond to Rl through R10. In some embodiments, the fluorophores that correspond to Rl through Rn are determined arbitrarily. For example, n is 10, and Rl corresponds to an Alexa 488 fluorophore, R2 corresponds to an Alexa 546 fluorophore, R3 corresponds to a 6-ROX (6-Carboxy-X-Rhodamine, or Rhodamine Red X) fluorophore, R4 corresponds to a Alexa Fluor 594, R5 corresponds to a Alexa Fluor 660, R6 corresponds to an Alexa 610 fluorophore, R7 corresponds to an Alexa 647 fluorophore, R8 corresponds to a3862735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924DyLight-510-LS fluorophore, R9 corresponds to an Alexa 405 fluorophore, and RIO corresponds to an Alex532 fluorophore. In some embodiments, other labels / fluorophores are used in the n-bit encoding system.

[0192] In some embodiments, the n-bit binary code is selected from the group consisting of 2- bit binary code, 3-bit binary code, 4-bit binary code, 5-bit binary code, 6-bit binary code, 7-bit binary code, 8-bit binary code, 9-bit binary code, 10-bit binary code, 11 -bit binary code, 12-bit binary code, 13-bit binary code, 14-bit binary code, 15-bit binary code, 16-bit binary code, 17-bit binary code, 18-bit binary code, 19-bit binary code, 20- bit binary code, 21 -bit binary code, 22-bit binary code, 23-bit binary code, 24-bit binary code, 25-bit binary code, 26-bit binary code, 27-bit binary code, 28 bit binary code, 29- bit binary code, and 30-bit binary code.

[0193] The methods and constructs described herein have significant advantages of those currently available in the art.EXAMPLES

[0194] EXAMPLE 1. Exemplary Method for Identifying Microbial Strains

[0195] The methods described herein can work as follows.

[0196] 1) Source material is obtained. Source material could be from patient / donor material(e.g. stool, inner mouth swab, vaginal swab), soil, food / agri cultural products, livestock, etc.

[0197] 2) Prepare sequencing library from source material. DNA or RNA is extracted from an aliquot of the source material and a sequencing library is prepared (using, for example, 16S amplification). Long read sequencing is performed on the library and 16S and / or 23 S sequencing data is used to create probes. Probes are designed from the sequencing data at the family, genus, species, or strain level depending on needs for community profiling and isolation. Probes can be prepared for IN-OUT detection (each encoding probe has readout probes attributed to a single color for fast screening) or standard HiPR-FISH barcode detection (using 11 unique fluorescent probes).

[0198] 3) Source material is prepared for culturing. Homogenize source / fecal material and create a 10-fold dilution series of replicate tubes in cryoprotectant and store at -80 °C. Replicate tubes can then be pulled from long term storage, and their contents pipetted on to agar for colony growth or dispersed into media in 96-well or 384-well plates for liquid growth. Media can be tailored to enrich, screen or select for desired growth properties. In the case of liquid growth, wells3962735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 are sealed and cultures can be monitored for growth by measuring changes in optical density until ready for harvest. To maximize the number of wells that are inoculated with a single viable cell and are therefore a pure culture, the method can aim for growth in 70 % of wells.

[0199] 4) Cultures are processed for short term storage, MALDI-ID and HiPR-ID. Wells of interest are identified, consolidated to ‘hit pick’ plates and the material is split for three downstream activities. A portion of each culture is (1) mixed with cryoprotectant and saved at -80 °C in 96 well plate formats or individual cryo tubes. (2) harvested, washed and transferred to MALDI chip with matrix additive, (3) fixed in 4% formaldehyde, followed by washing in PBS & resuspension in ethanol for HiPR-ID imaging.

[0200] 5) Cultures are analyzed with MALDI-ID. MALDI-TOF is performed across the hit pick plate to screen for known or previously isolated species vs. novel species or “unknown ID”.

[0201] 6) Cultures are analyzed with HiPR-ID. Fixed wells of interest are plated in a 96- well format (or on another array). HiPR-FISH (with an IN-OUT design panel or with a barcoded panel) is performed on the plate and a low-magnification objective on a widefield microscope is used to identify the wells of interest for inclusion.

[0202] 7) (optional) Wells or isolates of interest, which are positive in the IN-OUT design are re-assayed using the standard HiPR-FISH approach with a barcoded HiPR-ID panel to identify taxonomy (using confocal microscope and higher magnification to determine spectral barcodes).

[0203] 8) (as needed) Further isolation. When a species of interest is found to be part of a mixed-species culture, then the culture is retrieved from short term cryo storage plate, cultured on agar media using restreaking for isolation of a clonal population. When a pure culture of the desired strain is obtained, proceed to step 9.

[0204] 9) Proprietary isolate stock is made. Isolates identified for inclusion in the therapeutic strain bank are then retrieved from short term cryo storage plate, cultured and (1) mixed with cryoprotectant in matrix tubes for long term storage, (2) Prepped for whole genome sequencing to provide strain-level genetic detail on the isolate.

[0205] EXAMPLE 2. HiPR-ID Screening

[0206] The workflow of HiPR-ID is as follows: given a set of fixed samples to be screened, samples are deposited on an array (either manually or via a robot) to which they remain adhered during the assay. If needed, each sample is deposited on at least 2 replicate arrays. As desired, a pan-microbial stain or other stains can be applied to each array of samples. A probe panel is4062735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 hybridized to the arrays of samples and imaging is used to screen the array for samples of interest. To increase speed of screening, this first panel can be single-readout to highlight 1 or more taxa as the same readout; widefield, low-resolution, automated imaging across the array can be applied to increase speed of result relative to sample. If further information about the identified samples is desired, replicate arrays can be hybridized with n-bit barcoded probes and imaged at high- resolution to acquire multi-excitation spectra, and classification can be applied to show which of the targeted taxa are present in these samples, giving guidance for further isolation work. (FIG. 1A).

[0207] Results

[0208] As shown in FIG. IB, replicate arrays of a given sample, from cultures of source fecal material, were prepared, stained with DAPI to identify all microbes in the sample, and then hybridized with either a single-readout probe panel (top image) or a 2-bit barcode panel and classified (bottom image). The first panel highlighted the presence of a targeted taxon (red microbes) in the mix of taxa within the sample; the spectra of the taxa with the second panel corresponded to Thomasclavelia sp. (yellow microbes in the post-classification bottom image).

[0209] EXAMPLE 3. HIPR-ID-ENABLED ISOLATION

[0210] After designing a probe panel against a source sample, HiPR-FISH can be conducted on the baseline source sample to confirm presence of taxa of interest, their viability and metabolic activity (via rRNA intensity or use of viability stains), and to create a reference library of morphologies and expected associated spectra to be used during later stages of isolation. As a first step of an isolation campaign, HiPR-ID high-throughput screening can be applied to determine appropriate growth conditions that enrich for desired taxa to be isolated, to determine an appropriate level of dilution of the source sample for further culturing, and to screen prepared dilutions for ones containing taxa of interest at higher abundance. As a next stage of isolation, specified dilutions of the source sample and growth conditions can be used to create a large number of cultures which can be screened with HiPR-ID for growth of taxa of interest. If identified cultures are mixed, these can be further diluted, grown and screened with HiPR-ID and these steps can be repeated until the taxa of interest is confirmed to be isolated (FIG. 2A). Isolates can be preserved and banked with cryoprotectant, and characterized by genomic sequencing. The same workflow can be applied to growth of / from colonies as detailed below.

[0211] Results4162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0212] As shown in FIG. 2B, a portion of a fecal sample was diluted 1 : 10 and fixed without culturing, stained with DAPI, then hybridized with a HiPR-ID probe panel, demonstrating presence of a variety of taxa and the prevalence of taxa which would be hybridized by the probes.

[0213] FIG. 3A-3F show additional HiPR-ID representative results. Table A summarizes the results.

[0214] Here, all cultures originated from a human fecal sample, diluted and grown at varying growth conditions. HiPR-ID probes were designed from sequencing of the original sample to target specific taxa (genuses and / or species) which metagenomics had suggested were present in the sample, and used to screen cultures for these taxa of interest. The cultures were split into 3 portions: a live portion stored with cryoprotectant, a fixed portion for HiPR-ID, and a third portion used for MALDI-ID.

[0215] HiPR-ID identifies pure cultures. Here, HiPR-ID and MALDI on the same liquid culture predicted the same Bifidobacterium species. Upon restreak of a preserved glycerol stock of the culture, and growth of a clonal culture, whole genome sequencing confirmed the genus as Bifidobacterium, demonstrating that HiPR-ID can get the same result as MALDI and sequencing on pure cultures (FIG. 3 A).

[0216] HiPR-ID identifies low abundance taxa in mixed cultures.: This liquid culture gave a result of “not reliable identification” from MALDI. The sample was hybridized with HiPR-ID probes, imaged and classified; low-abundance Slackia sp. (light blue cells in image) were identified. A portion of the culture which had been cryopreserved was regrown and whole genome sequencing identified 1% as Slackia sp. (FIG. 3B).

[0217] HiPR-ID and imaging can give more information than MALDI: Here, HiPR-ID imaging showed a pure culture of Bifidobacterium sp., while MALDI of the same liquid culture gave a result “Not reliable identification,” suggesting that HiPR-ID is a more robust method than MALDI (FIG. 3C).

[0218] HiPR-ID can give a result with low biomass: On a liquid culture, MALDI gave a result of “No peaks found” corresponding to insufficient biomass for identification. HiPR-ID on a portion of the same culture showed sparse but identifiable microbes. Spectral imaging and analysis showed presence of a small round microbe not of interest (no panel signal) mixed with a rodshaped microbe with a signal suggesting a microbe in the family Oscillospiraceae, of either genus Ruthenibacterium or genus Lawsonibacter. A cryopreserved portion of the culture was streaked4262735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 on solid media; direct colony MALDI gave a result of “No ID possible.” A clonal culture was grown from another colony and sequenced, giving the result of Lawsonibacter sp. (FIG. 3D).

[0219] HiPR-ID enabled high-throughput screening: Here, 192 wells of cultures were screened at low magnification with probes for a single taxa and one well was identified as containing the taxa of interest, visible as green signal in FIG. 3E.

[0220] Morphology information from HiPR-ID workflow can guide isolation: Here, hybridization of HiPR-ID 2-bit barcoded probes to microbes from a well revealed a pure culture with one emissive readout, suggesting a taxa in or related to the taxa targeted by the panel, but no clear second readout signal to determine a barcode. Morphology comparison to reference images suggested Blautia sp. A portion of the culture which had been cryopreserved was streaked on solid media and MALDI was performed on a single colony, giving the result of Blautia sp. (FIG. 3F).

[0221] Table A. Summary of HiPR-ID Results

[0222] EXAMPLE 4. HIPR-ID ON POOLED SAMPLES4362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0223] Isolation of rare or difficult-to-culture taxa of interest can require screening a high number of samples; depending on the expected abundance of these taxa, samples can be pooled to increase throughput. For example, and as shown in FIG. 4A, if screening a well-plate of liquid cultures, pools of portions of cultures across each row or column can be easily prepared manually or robotically. Higher level pools of these pools or higher level pools directly from the plate, representing many more samples, can be prepared as well. The pools can be deposited on arrays and screened with HiPR-ID for taxa of interest; for each pool identified, the source pools or individual samples can then be screened with HiPR-ID and the process of screening sources can be repeated until the original source sample can be identified for further isolation.

[0224] Results

[0225] A pool of 8 cultures (a single column of a 96-well plate) was prepared and assayed with a barcoded probe panel. Imaging and classification revealed the presence of Erysipelotrichaceae in the mixed sample ((FIG. 4B, left, yellow microbes). The 8 source cultures were deposited separately onto an array and hybridized with the same barcoded probe panel; imaging and classification identified a single source well containing the Erysipelotrichaceae.

[0226] EXAMPLE 5. HIPR-ID FOR COLONY SCREENING

[0227] If colony growth is a desired strategy for a given taxa or isolation campaign, HiPR-ID can be applied to colonies as shown in FIG. 5A. An initial sample is cultured on solid media for growth, for example through streaking, spread-plating, or some other method. The live colonies can be picked from and directly deposited onto an array for imaging, with optional staining and / or HiPR-FISH to give more information. The picked colonies could also be deposited into separate containers for fixation (if multiple assays from the same colony are desired) prior to transfer to an array for imaging. Another method could include stamping an array onto the colonies directly, non-destructively sampling the space. After samples are adhered on the array, staining and optional hybridization with one or more probe panels can be followed by imaging to ID the locations of taxa of interest. Automated and fast imaging strategies could be employed to speed up the identification of colonies of interest and reduce manual effort; post-imaging computational strategies could be used to increase accuracy of location of colonies of interest relative to the original plate. Finally, the locations of colonies of interest would be mapped back to the original live plate and the live plate would be used for further isolation efforts.

[0228] Results.4462735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924

[0229] As shown in FIG. 5B, bacterial colonies were grown on agar via streaking. A glass slide was stamped onto the plate and colonies were seen to visibly adhere to the slide.

[0230] As shown in FIG. 5C, three species were struck out on agar, creating 3 streaks (the letters ‘K’, ‘A’, and ’N’, top left). A piece of tape was used to lift biomass off of the grown streaks and affixed to a slide (middle top). A HiPR-FISH assay was performed on the tape and a widefield tilescan revealed the expected layout of the streaks had been retained and was visible under fluorescent illumination (middle image). Confocal, high magnification images (below the tilescan) were analyzed and classified to reveal the expected species. (White objects indicate cells with the expected spectra; gray cells had either different spectra or did not pass area and classification thresholds.) Biomass from the “K” streak where E. coli was identified was re-streaked on a fresh agar plate (bottom left), and HiPR-FISH followed by confocal imaging and classification (rightmost images) revealed that the expected E. coli had been recovered, demonstrating the feasibility of isolation with this method.

[0231] EXAMPLE 5.1. REPLICA PLATING

[0232] In addition to the direct colony sampling approaches described in FIG. 6A, to reduce disturbance to the colonies and increase biomass available for further isolation, a replica plating strategy could also be employed, where one replica plate could be stored live and one could be used for screening (FIG. 6A). From the plate used for screening, direct fixation of colonies on the plate followed by the approaches described above could be employed to identify the locations of taxa of interest.

[0233] Results.

[0234] E. coli colonies were grown on agar via streaking. The plate was fixed; colonies were hybridized with FISH probes and then a slide was stamped onto the plate and imaged. Imaging, as shown in FIG. 6B, showed that microbes from the colonies had adhered to the slide and the microbes had the expected morphology & fluorescent signal from the probes.

[0235] EXAMPLE 6. REPLICA PLATING

[0236] The master plate was stamped onto a Whatman filter paper, secured on a custom 3-D printed stage; colony biomass visibly appeared on the filter paper. (FIG. 7B). A replica plate was created by stamping a fresh plate onto the filter paper (FIG. 7C). A slide was then stamped onto the filter paper and removed following colony transfer, then fixed and processed through HiPR- FISH. As shown in FIG. 7D, widefield tilescan in DAPI demonstrated colony transfer and the4562735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 classification results showed successful identification of the expected microbes in the different colonies via HiPR-FISH. Additionally, a mixed inoculum containing different taxa resulted in classification of multiple taxa, showing that the method could be used for mixed colonies (FIG. 7E, right). Meanwhile, the replica plate was regrown at 37°C, resulting in live colony replicates of the original plate which could be used in later downstream isolation efforts.

[0237] Details of one or more embodiments are set forth in the accompanying drawings and description. Other features, objects, and advantages will be apparent from the description, drawings, and claims. Although a number of embodiments of the invention have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. It should also be understood that the appended drawings are not necessarily to scale, presenting a somewhat simplified representation of various features and basic principles of the invention.4662735521 1

Claims

Attorney Docket No.: 273089 / KBS-007WO / 576924WHAT TS CLAIMED IS:

1. A method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) diluting the sample to obtain a diluted sample;(iii) dispersing the diluted sample into a multi-well plate; and(iv) analyzing the wells with a screen to identify microbial strains.

2. The method of claim 1, wherein prior to (iv) analyzing the wells, wells of interest are identified.

3. The method of claim 1 or claim 2, wherein all of the wells are analyzed in step (iv).

4. The method of claim 1 or claim 2, wherein a portion of the wells are analyzed in step (iv).

5. A method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate to obtain live cultures;(vi) optionally, identifying wells of interest with a first screen or growth and consolidating the wells of interest into at least one second multi-well plate; and (vii.A) analyzing the first multi-well plate or second multi-well plate with a first screen to identify microbial strains;(vii.B) analyzing the first multi-well plate or second multi-well plate with a second screen to identify microbial strains; and / or(vii.C) storing the live cultures of the first or second multi-well plate.4762735521 1Attorney Docket No.: 273089 / KBS-007WO / 5769246. The method of claim 5, wherein the first screen comprises assaying the first multi-well plate with a first identifying assay.

7. The method of claim 6, wherein the first identifying assay comprises:(A) optionally, fixing and permeabilizing a portion of the cultures from each well of interest or from a subset of wells of interest, and plating onto an array to obtain plated resuspensions, wherein the fixing and permeabilizing is done via resuspension of the cultures in ethanol;(B) providing a first set of probes, wherein the first set of probes comprises at least one first encoding probe and at least one first emissive readout probe;(C) contacting the first set of probes with the plated resuspensions to form a first complex;(D) imaging the array with a widefield microscope to acquire one emission spectra from the at least one first emissive readout probe; wherein the at least one first emissive readout probe emits a first color or a first set of colors, and wherein the first color or the first set of colors are assigned to a known microbrial strain.

8. The method of claim 5, wherein the method (vii.B) analyzes the first or second multi-well plate with a second screen to identify microbial strains.

9. The method of claim 8, wherein the second screen comprises (viii) performing a second identifying assay on the second multi-well plate, wherein the second identifying assay comprises:(A) optionally, fixing and permeabilizing a portion of the cultures from each well of interest or from a subset of wells of interest, and plating onto an array to obtain plated resuspensions, wherein the fixing and permeabilizing is done via resuspension of the cultures in ethanol;(B) providing a second set of probes,4862735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924 wherein the second set of probes comprises at least one second encoding probe and at least one second emissive readout probe;(C) contacting the second set of probes with the wells of interest to form a second complex;(D) imaging the array with a widefield or confocal microscope to acquire one emission spectra from the at least one second emissive readout probe; wherein the at least one second emissive readout probe emits a second color or a second set of colors; and wherein the second color or second set of colors are assigned to a known microbial strain.

10. The method of any one of claims 7-9, wherein the first set of encoding probes and second set of encoding probes are the same sequences.

11. The method of claim 5, wherein the method further comprises (ix.A) identifying wells of the first or second multi-well plate that emit the first or second color, or set of colors, to obtain generate a list of wells of interest.

12. The method of claim 5, wherein the method further comprises (ix.B) identifying wells of the first or second multi-well plate that do not emit the first or second color, or set of colors, to obtain generate a list of wells of interest.

13. The method of claim 5, wherein the method further comprises (ix.C) regrowing cultures from the stored live cultures of the first or second multi-well plate of step (vii.C).

14. The method of claim 5, wherein the method further (x) analyzes the first or second multiwell plate with a screen to identify microbial strains.

15. The method of claim 14, wherein the method continues the screen until one or more microbial strains of interests is confirmed to be isolated.

16. A method for identifying microbial strains from a sample, the method comprising:4962735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(i) providing a sample;(ii) optionally, homogenizing the sample;(iii) diluting the sample by at least 5-fold to obtain a diluted sample;(iv) dispersing the diluted sample into media in a first multi-well plate for liquid growth;(v) culturing the sample for at least a day in the first multi-well plate;(v.A) identifying wells of interest, wherein the wells of interest are wells with growth;(vi) pooling wells of interest to obtain a pooled sample, wherein the pooling comprises removing a portion of the wells of interest;(vii) plating the pooled sample on an array;(viii) identifying a pool of interest with a first screen;(ix) isolating the pool of interest and performing steps (iii)-(v) and (vii)-(viii) on the pool of interest;(x) performing a second screen to identify a culture of interest from the pool of interest;(xi) isolating the culture of interest and performing a third screen until a microbial strain of interest is identified.

17. The method of claim 16, wherein the first, second, and third screens are performed with an identifying assay, each screen comprising:(A) providing a set of probes, wherein the set of probes comprises at least one encoding probe and at least one emissive readout probe;(B) contacting the set of probes with the pool of interest or wells of interest to form a complex; and(C) imaging the array with a widefield microscope to acquire one emission spectra from the at least one emissive readout probe; wherein the at least one emissive readout probe emits a color and wherein the color is assigned to a pool of interest, well of interest, or microbial strain.

18. A method for identifying microbial strains from a sample, the method comprising:5062735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(i) providing a sample;(ii) culturing the sample on solid media to provide colonies or biomass;(iii.A) picking live colonies and depositing the live colonies on an array; or(iii.B) picking live colonies and depositing the live colonies on a container for fixation; then transferring the fixed colonies to an array; or(iii.C) stamping an array onto the live colonies directly;(iii.C.l) optionally, fixing the colonies on the array;(iv) staining the array of any of steps (iii. A)-(iii.C);(v) performing a screen on the array of any of steps (iii.A)-(iii.C) to identify microbial strains of interest;(vi) optionally, repeating steps (iii.A)-(v); and(vii) optionally, re-culturing the microbial strain of interest from the plate in step (ii) and repeat steps (iii)-(v).

19. A method for identifying microbial strains from a sample, the method comprising:(i) providing a sample;(ii) culturing the sample on solid media to provide colonies;(iii) stamping a first array onto the live colonies directly;(iv) stamping a second array onto the first array;(v) generating at least one replica plate by stamping the first array onto a new plate;(vi) fixing and staining the second array;(vii) performing a screen on the second array to identify microbial strains of interest;(viii) optionally, repeating steps (iii)-(v); and(ix) optionally, re-culturing the microbial strain of interest and repeat steps (ii)-(v).

20. The method of any one of claims 1-19, wherein prior to identifying the wells of interest or pools of interest a pan-microbial stain is added.

21. The method of any one of claims 1-20, wherein prior to performing the first screen a reference panel is prepared.

22. The method of claim 21, wherein the reference panel is prepared by:5162735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924(i) extracting DNA or RNA from an aliquot of the sample to obtain a sequencing panel;(ii) performing long read sequencing on the sequencing panel to obtain RNA sequencing data; and(iii) designing encoding probes based on the obtained RNA sequencing data, wherein each encoding probe comprises a targeting sequence; and(iv) selecting the encoding probes to reflect the reference panel.

23. The method of any one of claims 1-22, wherein the sample comprises at least one cell, a cell suspension, a tissue biopsy, a tissue specimen, bone biopsies, organoids, three-dimensional hydrogel scaffolds, transwell systems, or plant biopsies.

24. The method of any one of claims 1-23, wherein the sample is a cell; optionally, wherein the cell is a bacterial or eukaryotic cell.

25. The method of any one of claims 1-24, wherein the sample is a stool sample from a human donor.

26. The method of any one of claims 1-25 wherein the microbial strain is a strain derived from a microbe selected from the group consisting of a bacterium, a synthetic bacterium, a synthetic organism, a fungus, a virus, an archaea, a parasite, and a genetically modified organism.

27. The method of claim 26, wherein the microbial strain is a bacterial strain.

28. The method of any one of claims 1-27, wherein the microbial strain is a genetically engineered strain; optionally, wherein the genetically engineered strain is a genetically engineered microbial strain.

29. The method of any one of claims 1-28, wherein each of the encoding probes comprise a targeting sequence.5262735521 1Attorney Docket No.: 273089 / KBS-007WO / 57692430. The method of claim 29, wherein the targeting sequence targets at least one of messenger RNA (mRNA), micro RNA (miRNA), long non-coding RNA (IncRNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), transfer RNA (tRNA), Crispr RNA (crRNA), transactivating cirspr RNA (tracrRNA), mitochondria RNA, Intronic RNA, viral mRNA, viral genomic RNA, environmental RNA, double-stranded RNA (dsRNA), small nuclear RNA (snRNA), small nucleolar (snoRNA), piwi-interacting RNA (piRNA), genomic DNA, synthetic DNA, DNA, plasmid DNA, a plasmid, viral DNA, retroviral DNA, environmental DNA, extracellular DNA, a protein, a small molecule, or an antigenic target.

31. The method of claim 30, wherein the targeting sequence targets mRNA and / or rRNA.

32. The method of any one of claims 29-31, wherein(a) the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23 S rRNA sequence in the microbial strain; and / or(b) the targeting sequence targets a 16S rRNA sequence, a 5S rRNA sequence, and / or a 23 S rRNA sequence in the pathogen.

33. The method of any one of claims 29-31, wherein the targeting sequence targets a 18S rRNA sequence, a 5.8S rRNA sequence, and / or a 28S rRNA sequence in the in the microbial strain, wherein the microbial strain is a eukaryotic microbial strain.

34. The method of any one of claims 1-33, wherein the emissive readout probe comprises a label on the 5’ and / or 3’ end.

35. The method of claim 34, wherein the label is Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647-R-phycoerythrin, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-allophycocyanin, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Alexa Fluor Plus 405, Alexa Fluor Plus 488, Alexa Fluor Plus 555, Alexa Fluor Plus 594, Alexa Fluor Plus 647, Alexa Fluor Plus 680, Alexa Fluor Plus 750, Alexa Fluor Plus 800, Pacific Blue, Pacific Green, Rhodamine Red X, DyLight 485-LS, DyLight-510-LS, DyLight 515-LS,5362735521 1Attorney Docket No.: 273089 / KBS-007WO / 576924DyLight 521 -LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Fluorescein FITC, R- phycoerythrin (PE), Tamara, Cy3.5 581 , Rox, Red 613, Texas Red, Cy5, Cy5.5, Cy7, Allophycocyanin, ATTO 430LS, ATTO 490LS, ATTO 390, ATTO 425, Cyan 500 NHS-Ester, ATTO 465, ATTO 488, ATTO 495, ATTO Rhol 10, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 643, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740.

36. The method of any one of claims 1-35, wherein the emissive readout probes are imaged using widefield epifluorescence microscopy, widefield microscopy, point scanning confocal microscopy, spinning disk confocal microscopy, lattice lightsheet microscopy, or light field microscopy.

37. The method of claim 36, wherein the detection strategy used is channel, spectral, channel and fluorescence lifetime, or spectral and fluorescence lifetime.

38. The method of any one of claims 1-37, wherein the array is a microscope slide, at least one chamber, at least one microfluidic device, at least one well, at least one plate, at least one filter membrane, or at least one adhesive tape.

39. The method of any one of claims 1-38, wherein the sample is diluted by at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 100-fold, at least a 1000-fold, at least a 10,000-fold, or at least a 100,000-fold.

40. The method of any one of claims 1-39, wherein the sample is cultured until growth is detected in at least 51%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the wells of the first multi-well plate.

41. The method of any one of claims 1-39, wherein the sample is cultured for two days.5462735521 1Attorney Docket No.: 273089 / KBS-007WO / 57692442. The method of any one of claims 1-39, wherein the sample is cultured as growth is detected in groups of wells from the first multi-well plate.

43. The method of any one of claims 1-42, wherein the first multi -well plate is a 6-well plate, 8-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, or a 1536- well plate.

44. The method of any one of claims 1-43, wherein the second plate is a single-well plate or a multi-well plate.

45. The method of claim 44, wherein the second multi-well plate is a 6-well plate, 8-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, or a 1536-well plate.

46. The method of any one of claims 1-45, further comprising, isolating the identified microbial species and incorporating into a strain bank.

47. The method of claim 46, wherein whole genome sequencing is performed on the isolated microbial species to obtain strain-level genetic information.

48. The method of claim 47, wherein synthetic strains are prepared based on the genetic information obtained by the whole genome sequencing.5562735521 1

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