JP2019531730A - Bacterial community relationship determination and other high-throughput microbiology applied high resolution systems, kits, devices, and methods - Google Patents

Abstract

A method is provided for analyzing a sample comprising a population of biological entities using at least one microfabricated device. Each of the plurality of microwells on the microfabricated device is uniquely indexed and the sample is filled such that at least some of the microwells each contain two or more cells of the biological entity. The microfabricated device is incubated under predetermined conditions, and the selected genetic material of the biological entity cells obtained from the incubation is amplified to obtain an amplicon. Sequence amplicon collections, obtain sequencing data, and based on that and based on microwell indexing, obtain identification of biological entities present in each of the plurality of microwells . Such identification may be used to determine relationships between different types of biological entities in the sample.

Description

  This application is within the scope of the claims of 15 / 135,377 US continuation application filed on April 21, 2016 and 62 / 400,841 US provisional application filed September 28, 2016. Claims the priority of each of these applications, the entire text of each of which is incorporated herein by reference.

[Sequence Listing]
This application includes a 3 KB size sequence list submitted in ASCII format via EFS-Web created on September 27, 2017 and named “GALT — 007_PCT_ST25.txt”. The entire contents of the sequence list to be incorporated are incorporated herein by reference.

  The present invention relates generally to innovations in microbiology, microfabrication, chemistry, optics, robotics and information technology. More specifically, the present invention relates to high throughput culture, sorting, isolation, sampling and / or identification systems, devices, kits and methods of biological entities and / or nutrients.

  Prior art techniques and means for culturing biological entities from the natural environment and other samples are often slow, labor intensive, time consuming and expensive. Even with these conventional techniques and means, cells and other biological entities often still refuse any attempt to culture and miss the opportunity to obtain information and / or products. Similarly, screening a group of biological entities for specific metabolites, enzymes, proteins, nucleic acids, phenotypes, mutations, metabolic pathways, genes, adaptability, performance and / or therapeutic benefits can be challenging. Need a complex and effective method. For example, microorganisms live in highly dangerous environments. To survive, microorganisms come from a surprising set of biochemical means, including new enzymes, unique metabolites, innovative genetic pathways, and manipulation strategies to the environment and neighboring microorganisms: new insights and life-saving antibiotics. It has created a powerful solution that could lead to products up to fertilizers that improve food production and safety.

  The present invention provides microbiology systems, devices, kits and methods that streamline the culture workflow, assist in high-throughput sorting, and / or create new pathology and products according to embodiments.

  In embodiments, a method for analyzing a sample comprising a population of biological entities is provided. This method can use at least one microfabricated device having a defined (or included) top surface of the microwell array, and the microwell array is unique to a plurality of microwell arrays. (1) a target-specific nucleic acid sequence for annealing a nucleic acid fragment without the target of one or more biological entities present in the microwell ) And (2) contains a position-specific nucleotide sequence for identifying the position of the microwell. The method includes loading a sample onto a microfabricated device such that at least some of the plurality of microwells each contain one or more cellular biological entities and a certain amount of nutrients, respectively. And incubating the microfabricated device under predetermined conditions, and within individual microwells, selected genetic material of cells of a biological entity obtained from incubation in a plurality of microwells (eg, The first amplicon in at least a subset of the plurality of microwells, and thereby sequencing the first amplicon collection collected from the subset of the plurality of microwells Sequencing data to obtain the sequencing data and the fixed data contained in each of the plurality of microwells. Of based on the tag nucleic acid molecule, to obtain an identification of biological entities present in each of the subset of the plurality of micro-wells of the at least one micromachined device.

  The methods shown in embodiments determine the presence or absence of a sample relationship between at least two types of biological entities based on the identification of biological entities present in each of the plurality of subsets of microwells. To do. The relationship may be any of a dependent relationship, a symbiotic relationship, or a destructive relationship. In embodiments, the presence or absence of a relationship may be determined by comparing across multiple microwells what type of biological entity is not present in the same microwell after incubation.

  In some embodiments, the method includes: N samples of a biological entity averaged in each microwell of a plurality of microwells (N cells are of the same type or N may be two or more. For example, N can take a number between 2 and 100, between 2 and 50, and between 2 and 10.

  In the methods shown in embodiments, the biological entity in the sample comprises bacteria. The bacteria may include bacteria of different strains, different species or different genera. The population of biological entities in the sample may include microorganisms that are naturally present in a particular environment. For example, the biological entity may be a collection of microorganisms obtained from human stool, human intestine, human skin, human nasal cavity, vagina, soil, rhizosphere, water, and the like. In other embodiments, the biological entity in the sample may include viruses, fungi, or eukaryotic cells.

  The methods shown in embodiments may apply a membrane to the top surface of a microfabricated device to retain a biological entity filled across multiple microwells prior to incubation. Nutrients may be pre-filled in microwells and retained by a membrane. The nutrient may include a plurality of different nutrients filled across the microwells of at least one microfabricated device. In such a case, determining the presence or absence of a relationship between at least two types of biological entities may include determining such a relationship depending on different nutrients. In embodiments, nutrients may be included in a reservoir provided on the membrane and outside the microwell. In such cases, the membrane may be permeable to nutrients and the nutrients may be movable from the reservoir through the membrane to the microwell.

The methods shown in embodiments may further transfer at least some of the cells of the biological entity obtained from the incubation to the target location. In such cases, amplification and sequencing may be performed on any cells that have not been transferred to the target location or cells that have been transferred to the target location.
In some embodiments, the unique tag nucleic acid molecule in each of the plurality of microwells forms part of the PCR primer used for amplification.

In some embodiments, the surface density of at least one microwell array of microfabricated devices is at least 150 per micro well cm ^2, at least 250 microwells per cm ^2, cm ² per at least 400 microwells per cm ² At least 500 microwells cm ² per at least 750 microwells cm ² per at least 1,000 microwells cm ² per least 2,500 microwells cm ² per least 7,500 microwells at least 10,000 per micro well cm ^2, cm ² per at least 50,000 microwells cm ² per 100,000 microwells, or cm ² per may have a surface density of at least 160,000 microwells. In some embodiments, each microwell of at least one microfabricated device microwell may have a diameter of about 5 μm to about 500 μm, about 10 μm to about 300 μm, or about 20 μm to about 200 μm.

  The methods shown in embodiments provide a method for analyzing a sample comprising a population of biological entities using at least one microfabricated device having a top surface defining an array of microwells. The method comprises: (1) a target-specific nucleotide sequence for annealing to a target nucleic acid fragment of one or more biological entities, and (2) a microwell in each of a plurality of microwells of a microwell array A position-specific nucleotide sequence for identifying the location of at least one of the plurality of microwells, each of which is microfabricated to contain one or more cells of a biological entity and a certain amount of nutrients Loading of sample onto device; Incubation of microfabricated device at predetermined conditions; Selection of cells of biological entity obtained from incubation in at least a subset of a plurality of microwells of at least one microfabricated device Amplification of the generated genetic material, resulting in multiple A first amplicon in the clowell; sequencing a first amplicon collection collected from a subset of the plurality of microwells to obtain sequencing data; and sequencing data and a plurality of microwells Filling with a unique tag nucleic acid molecule comprising obtaining an identification of a biological entity present in each of a plurality of microwell subsets of at least one microfabricated device based on the unique tag nucleic acid molecule contained in each including.

  In embodiments, a method for analyzing a microbiome of microorganisms collected from a particular environment is provided. The method uses at least one microfabricated device having a top surface defining an array of microwells, each of the plurality of microwells of the array of microwells being (1) a target specific nucleotide sequence for annealing microwells A unique tag nucleic acid molecule comprising a target nucleic acid fragment of one or more biological entities of interest that may be present in, and (2) a position-specific nucleotide sequence for identifying the location of a microwell on a microfabricated device including. In this method, a sample prepared from a microbiome is loaded onto at least one microfabricated device so that each microwell has an average of 2-20 cells of microbiome and an amount of nutrients on average. Including; incubating the microfabricated device under predetermined conditions; amplifying selected genetic material of the microorganism resulting from the incubation in at least a subset of the plurality of microwells in the at least one microfabricated device, as a result Obtaining a first amplicon in a plurality of microwells; sequencing a first amplicon aggregate collected from a subset of the plurality of microwells to obtain sequencing data; and sequencing data and Unique tag in each of multiple microwells Based on acid molecules, obtaining identification of microorganisms present in each of the subsets of the plurality of microwells of the at least one microfabricated device; and in the microbiome based on identification of microorganisms present in the plurality of microwells Determining the presence or absence of a relationship between at least two different types of microorganisms.

  In embodiments, a method is provided for analyzing relationships between microbiome microorganisms collected from a particular environment. The method consists of: separating microbiome microorganisms each containing on average 2 to 20 cells into a plurality of parts; separating each part of the plurality of parts of the microorganisms in separate compartments in the presence of nutrients under predetermined conditions Incubation; after incubation, identification of microorganisms present in each of at least a subset of the isolated compartments; and based on a comparison across the subset of compartments whether the identified microorganism type is present or absent in the same microwell Including the determination of the presence or absence of a relationship between at least two different types of microorganisms in the microbiome.

  It should be appreciated that all combinations of the above concepts and those described in more detail below (as long as they do not contradict each other) are considered as part of the aggregate subject matter disclosed herein. . In particular, all combinations of subject matter recited in the claims presented at the end of this disclosure are contemplated herein as part of the aggregate subject matter of the disclosure. It is also reasonably appreciated that the terms used in any disclosure expressly used herein and incorporated by reference should be given the meaning most consistent with the particular concept of the disclosure. It is.

  Other systems, processes and functions will become apparent to those skilled in the art upon review of the following drawings and detailed description. All such additional systems, processes and functions are intended to be encompassed within the specification of the invention, fall within the scope of the invention, and be protected by the accompanying claims.

  It should be understood by those skilled in the art that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale, and in some instances, various aspects of the inventive subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate understanding of different mechanisms. In the drawings, like reference numbers generally indicate identical features (eg, functionally similar and / or structurally similar components).

  FIG. 1 is a perspective view illustrating a microfabricated device or chip according to embodiments.

  2A-2C are top, side, and end views, respectively, illustrating the size of a microfabricated device or chip according to multiple embodiments.

  FIGS. 3A and 3B are an exploded view and a top view, respectively, illustrating a microfabricated device or chip according to embodiments.

  4A and 4B are illustrations of membranes according to multiple embodiments. FIG. 4C is an image of a membrane surface with an impression formed from contact with an array of wells according to embodiments.

  FIG. 5A is an operational step diagram illustrating a method for isolating a number of cells from a sample according to embodiments. FIG. 5B is an illustration of a method of isolating a number of cells from a soil sample according to embodiments.

  FIG. 6 is an operational step diagram illustrating a method for isolating and culturing a number of cells from a sample according to embodiments.

  FIG. 7 is an illustration of a method for isolating and culturing a number of cells from a composite sample according to embodiments. Panel 716 shows the output of an isolated cultured cell line (SEQ ID NO: 2-6).

  FIG. 8A to FIG. 8C are explanatory diagrams of sampling by one pin or multiple pins performed according to a plurality of embodiments.

  9A-9D are images showing selection of one well according to embodiments.

  10A-10D are illustrations of a tool for harvesting from one chip according to embodiments.

  FIG. 11 is an image of a well punched through a thin layer of agar, illustrating the penetration of a membrane or sealing layer according to embodiments.

  FIG. 12 is a diagram illustrating a cross section of a chip 1200 according to a plurality of embodiments.

  FIG. 13 is a work step diagram illustrating a screening method according to a plurality of embodiments.

  FIG. 14 is a list diagram illustrating a screening method according to a plurality of embodiments.

  FIG. 15 is a series of images illustrating one screening example according to embodiments.

  FIGS. 16A-16C are images that illustrate collection from a single shield according to embodiments.

  FIG. 17A is an exploded view illustrating a sorting chip according to a plurality of embodiments. FIG. 17B is a fluorescence image of the chip following sorting according to embodiments. FIG. 17C is an image showing one step of collecting a sample from a chip following sorting according to embodiments.

  FIG. 18 is a work step diagram illustrating a counting method according to a plurality of embodiments.

  FIG. 19 is a list diagram illustrating a counting method according to a plurality of embodiments. Panel 1916 shows the output of cultured cell sequence and relative abundance (SEQ ID NO: 2-6).

  FIG. 20 is a list diagram illustrating an indexing method according to embodiments.

  FIGS. 21A-21E are lists illustrating chips having well-specific chemical agents according to embodiments.

FIG. 22 outlines the number of recent genera in the microwells of microfabricated devices according to embodiments.
Detailed description

  The present invention relates generally to systems, kits, devices and methods for the isolation, culture, adaptation, sampling and / or sorting of biological entities and / or nutrients. Disclosed is a microfabricated device (or “chip”) for containing a sample containing at least one biological entity. The term “biological entity” may include microorganisms, cells, cellular components, cell products, viruses and others, and the term “species” is used to describe a unit of classification and is Taxonomic units (OTU), genotypes, phylogenetics, phenotypes, ecotypes, provenances, behaviors or interactions, products, varieties, evolutionary significant species and others.

  The cells can be archaea, bacteria, eukaryotes (eg fungi) or mammalian cells. For example, the cell can be a microorganism such as an aerobic, anaerobic or a conditional aerobic microorganism. The virus can be a bacteriophage. Other cellular components / products include proteins, amino acids, enzymes, sugars, adenosine triphosphate (ATP), lipids, nucleic acids (eg DNA and RNA), nucleosides, nucleotides, cell membranes / walls, flagella, pili, organelles, It can include metabolites, vitamins, hormones, neurotransmitters, antibodies and others.

  Nutrients may be defined (eg, chemically defined or synthetic media) or undefined (eg, basal or complex media). Nutrients may include or be a component of laboratory formulated and / or commercially produced media (eg, a mixture of two or more chemicals). The nutrient may be a liquid nutrient medium (ie, nutrient soup) or a component thereof, such as marine broth, lysogenic broth (eg, luria broth), and the like. The nutrient may be a commercially available agar plate such as a liquid medium and / or a blood agar medium that is mixed with agar to form a solid medium, or may be a component thereof.

  The nutrient may include a selective medium or one component thereof. For example, the selective medium can be used for the growth of only certain biological entities or only biological entities that have certain properties (eg, antibiotic resistance or specific metabolite synthesis). Nutrients take advantage of biological properties in the presence of a specific indicator (eg, neutral red, phenol red, eosin Y, or methylene blue) to replace one type of biological entity with another type of biological entity. Alternatively, it may contain or be a component of an identification medium that distinguishes it from other biological entities.

  The nutrient may include a natural environment extract or a medium derived from the natural environment, or may be a component thereof. For example, nutrients can be derived from a natural environment, a separate environment, or multiple environments for a particular type of biological entity. The environment can be one or more biological tissues (eg, connective tissue, muscles, nerves, epithelium, plant epidermis, vascular bundles, soil, etc.), biological fluids or other biological products (eg, amniotic fluid, bile, blood, cerebrospinal cord) Fluid, earwax, exudate, feces, stomach fluid, interstitial fluid, intracellular fluid, lymph fluid, milk, nasal discharge, rumen contents, saliva, sebum, semen, sweat, urine, vaginal discharge, vomiting, etc.), microorganism suspension Suspension, air (eg, with various gas contents), supercritical carbon dioxide, soil (eg, with minerals, organics, gases, liquids, microorganisms, etc.), sediment (eg, agriculture, oceans) Sediment, etc.), living organic matter (eg, plants, insects, other small organisms and microorganisms), dead organic matter, horse grass (eg, pasture, legumes, storage grass, crop residues, etc.), minerals, Oil or oil product (eg animal, plant, petrochemical) Scientific origin), water (eg, natural fresh water, drinking water, sea water, etc.) and / or sewage (eg, sanitary, commercial, industrial and / or agricultural wastewater, and surface effluent), and others .

The microfabricated device can define a high density array of microwells for culturing at least one biological entity. The term “dense” can mean the ability of a system or method to allocate many experiments within a certain area. For example, a microfabricated device with a “high density” experimental unit can incorporate from about 150 microwells per cm ^{2 to} about 160,000 or more eyecrowells per cm ² as further described herein. Additional examples are shown in Table 1.

  Microfabricated devices can incorporate a substrate with a series of functional layers. The series of functional layers defines a first functional layer defining a first array of experimental units (eg, wells) and a next array of experimental units (eg, microwells) within each experimental unit of the preceding functional layer. At least one subsequent functional layer. Each of the experimental units can be configured to contain and culture and / or sort biological entities and / or nutrients. In particular, the systems, kits, devices and methods described herein can be used for automated and / or high-throughput sorting of various conditions on a dense matrix of cells. For example, the systems, kits, devices and methods described herein can examine and compare the effects of one or more different nutrients on microbial growth and / or test for metabolites, enzyme activity, mutations or other cellular functions. Can be used to

  FIG. 1 is a perspective view illustrating a microfabricated device or chip according to a plurality of embodiments. The chip 100 incorporates a base material formed in the form of a microscope slide glass having an injection molding mechanism on the upper surface 102. These mechanisms are four separate microwell arrays (or microarrays), etc. in addition to the protruding tag 106. The microwells in each microarray are arranged in a lattice pattern with the periphery of the chip 100 and the edges without wells between the microarrays 104.

  2A-2C are top, side, and end views, respectively, illustrating the size of the chip 100 according to multiple embodiments. In FIG. 2A, the upper surface of the chip 100 is approximately 25.5 mm × 75.5 mm. In FIG. 2B, the end surface of the chip 100 is approximately 25.5 mm × 0.8 mm. In FIG. 2C, the side surface of the chip 100 is approximately 75.5 mm × 0.8 mm.

  After loading the sample into the microfabricated device, a film may be applied to at least a portion of the device. FIG. 3A is an exploded view showing a microfabricated device 300 according to embodiments as viewed from the top of FIG. 3B. The device 300 incorporates a chip with a well array 302 containing, for example, soil microorganisms. A membrane 304 is placed on the top surface of the well array 302. A gasket 306 is placed on top of the membrane 304. A polycarbonate cover 308 with a fill hole 310 is placed on the upper surface of the gasket 306. Finally, the sealing tape 312 is attached to the cover 308.

  At least a portion of a microfabricated device that incorporates one or more experimental units, wells or microwells can be covered with a membrane. For example, after loading a sample into a microfabricated device, at least one film can be applied to at least one microwell of a microwell high density array. Multiple films can be attached to multiple portions of a microfabricated device. For example, separate films can be affixed to a plurality of different compartments of a microwell high density array.

  The membrane is tied to, attached, partially attached, affixed, and sealed to a microfabricated device to retain at least one biological entity in at least one microwell of a microwell high density array. And / or may be partially sealed. For example, the film may be reversibly applied to a microfabricated device by lamination. To place at least one biological entity into at least one microwell of a microwell high density array, the membrane is stamped, peeled, detached, partially detached, removed and / or partially removed Can be done.

  A portion of the cell population within at least one experimental unit, well or microwell may be attached to the membrane (eg, via adsorption). When attached, the cell population in at least one experimental unit, well or microwell pulls the membrane while a portion of the cell solid group in at least one experimental unit, well or microwell remains attached to the membrane. It can be sampled by peeling off.

  4A and 4B are illustrations of membranes according to multiple embodiments. FIG. 4A shows a side view of a chip 400 that defines a well array filled with contents and a membrane 402 that covers the well array and encapsulates the chip 400, and thus the surface of the membrane 402 in contact with the chip 400. Has an imprint of each of the wells with well content samples attached (eg, attached) thereto when peeled from the chip 400, as shown in FIG. 4B. FIG. 4C is an image of a stamped membrane surface formed by contact with a well array according to embodiments.

  The membrane is impermeable, semi-permeable, selectively permeable, differentially permeable, and / or partially permeable allowing at least one nutrient to diffuse into at least one microwell of the microwell high density array. Can be sex. For example, the membrane may be composed of natural and / or synthetic materials. The membrane may incorporate a hydrogel layer and / or filter paper. In embodiments, a membrane is selected that has pores that are small enough to hold at least some or all of the cells in the microwell. For mammalian cells, the pore size can be a few microns and still retain the cells. However, in embodiments, the pore size may be about 0.2 μm or less, such as 0.1 μm. The membrane diameter and pore size depend on the material. For example, a hydrophilic polycarbonate membrane can be utilized, for which the diameter can range from about 10 mm to about 3000 mm, and the pore size can range from about 0.01 μm to about 30.0 μm. An impermeable membrane has a pore size approximately equal to zero. In an embodiment using an impermeable membrane, all nutrients must be supplied to the microwells prior to sealing with the membrane. A membrane that is gas permeable but not liquid permeable can accept oxygen into the microwell and carbon dioxide from the microwell. The membrane may have a complex structure that may or may not have a limited pore size. However, the pores can be on the order of nanometers. Other factors in selecting a membrane may include cost, sealing performance and / or sterilization performance.

  The substrate can define a microwell array that is extended from a first surface to a second surface facing the first surface. The microchannel may have a first opening on the first surface and a second opening on the second surface. The first membrane can be applied to at least a portion of the first surface such that at least some of the cell population in the at least one microchannel is attached to the first surface. The second removable membrane can be affixed to at least a portion of the second surface such that at least some of the cell population within the at least one microchannel is attached to the second surface. The cell solid groups in the at least one microchannel may include cells in the first membrane and / or cells in the at least one microchannel while at least some of the cell solid groups in the at least one microchannel remain attached to the first membrane. Samples can be sampled by tearing off the second membrane while at least some of the solid groups remain attached to the second membrane.

  The term “high throughput” can mean the ability to allow a very large number of experiments that a system or method has to be performed quickly in parallel or sequentially. An example of a “high-throughput” system is that, as described herein, one or more biological entities, at least one metabolite, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, gene, adaptation Automated devices equipped with cell biology techniques for preparation, culture and / or numerous chemical, genetic, pharmaceutical, optical and / or image analysis can be included to test for type and ability. According to embodiments, “high throughput” can mean performing experiments on a scale ranging from at least about 96 experiments to at least about 10,000,000 experiments simultaneously or nearly simultaneously.

  The systems, devices, kits and methods disclosed herein can be used for high-throughput sorting of various conditions against a matrix of biological entities (eg, cells). The “in-well well” concept refers to any level required or desired by manufacturing (eg, microfabricating) a substrate or chip to have multiple levels of functional layers (ie, wells in wells, wells in wells, etc. ) Can also be realized. The first functional layer can define an experimental unit (eg, well) array. Each experimental unit provides another functional layer by defining the next experimental unit (eg, microwell) array. As a result, multiple experiments or tests can be performed simultaneously on a single chip, thereby enabling high-throughput operation.

For example, in FIGS. 3A and 3B, a gasket 306 is placed on top of the membrane 304, which membrane is applied to the well array 302 of the microfabricated device 300 according to embodiments. The gasket 306 has only one opening. However, in a further embodiment, multiple smaller gaskets with one smaller opening or a single gasket with multiple smaller openings are placed on the top surface of the device (with or without a membrane), Thereby, a step of forming a functional layer, or an array of larger experimental units with the next functional layer, or the next experimental unit array (e.g., well 302) located therein may be formed.
With multiple levels of functional layers, for example, multiple nutrients or nutrient formulations can be tested simultaneously or nearly simultaneously. The same scheme can be used, for example, to test for metabolite or cell specific functions or to separate a microorganism from a natural environment derived nutrient into another nutrient.

  The experimental unit is a predetermined part on the surface of the microfabricated device. For example, the surface of the chip can be designed to immobilize cells in a first array of predetermined sites. These predetermined sites can be wells, microwells, microchannels and / or designated immobilization sites. For example, the surface may be microfabricated so as to define a microwell array. The array can be divided into several sections by the step of defining walls or additional walls in the substrate. For example, the surface may be microfabricated such that it first defines a first array of wells, such that the internal surface of each well defines a second array of microwells, microchannels or immobilization sites. Are processed in order. In another example, the surface can be processed to define a microwell array, and another substrate (eg, agar, plastic or other material) is defined by the surface and thereby It can be applied to the surface of the microwell to partition it. Each well, microwell, microchannel and / or immobilization site can be constructed to contain and grow at least one cell, but in use, whichever well, microwell, microchannel or immobilization site In fact, one or more cells may or may not be accommodated and / or grown. The experimental unit type may be interchangeable. For example, embodiments in this document that specifically describe microwells also disclose embodiments in which those microwells are at least partially replaced with microchannels, immobilization sites, and / or other types of experimental units. Also intended.

  One or more portions of the microfabricated device can be selected, treated, and / or applied with a surface chemistry modifier to have a special surface chemistry. For example, at least a portion of the substrate surface has a first surface property that does not attract cells and / or reduces the tendency to stick to the surface of cells or a second tendency to increase the tendency to attract and / or stick cells to the surface of cells. Can be designed with various surface properties. Depending on the type of target cell, the substance and / or coating can be hydrophobic and / or hydrophilic. At least a portion of the top surface of the substrate may be treated to have a first surface property that does not attract the target cells and / or reduces the tendency of the target cells to stick to that surface. On the other hand, at least a portion of the internal surface of each experimental unit, well or microwell has a second property that attracts the target cells and increases the tendency of the target cells to occupy the experimental units, wells or microwells, May be processed. One surface of the substrate may have multiple portions with different surface characteristics.

  The surface chemical modifier can be applied using chemical vapor deposition, electroporation, plasma treatment and / or electrolytic deposition. The surface chemical modifier can control surface potential, lund potential, zeta potential, surface morphology, hydrophobicity and / or hydrophilicity. The surface chemical modifier may include silane, polyelectrolyte, metal, polymer, antibody and / or plasma. For example, the surface chemical modifier may be octadecyltrichlorosilane or the like. The surface chemical modifier is a dynamic copolymer such as polyoxyethylene (20) sorbitan monolaurate and / or polyethylene glycol p- (1,1,3,3-tetramethylbutyl) -phenyl ether. Can be included. The surface chemical modifier may comprise a static copolymer such as, for example, poloxamer 407, poly (L-lysine) and / or poly (ethylene glycol) -poly (1-lysine) block copolymer.

  An apparatus for sorting various conditions against a matrix of cells can include a substrate having a surface that defines a microwell array. Several sections of the microwell array can be divided into subarrays (eg, by larger wells or walls). The substrate can be made by microfabrication. Each microwell can contain and grow a biological entity (eg, a cell). The resulting matrix of biological entities (eg, cells) can be a dense matrix of biological entities. The first array and / or the second array can be planar, generally planar, and / or multi-planar (eg, on a roll).

  The term “high resolution” can mean the ability of a system or method to distinguish between a number of available experiments. For example, a “high resolution” system or method can select one experimental unit having a diameter of about 1 nm to about 800 μm from a microfabricated device containing a high density experimental unit. The substrate of the microfabricated device or chip can incorporate about 10,000,000 or more microwells. For example, a microwell array can include at least 96, at least 1,000, at least 5,000, at least 10,000, at least 100,000, at least 500,000, at least 1,000,000, at least 5,000,000, or 10,000,000 storage locations.

The surface density of the microwells can be from about 500 microwells to about 160,000 microwells per cm ² or more. The substrate microfabricated device or chip, cm ² per at least 150 microwells cm ² per at least 250 microwells cm ² per at least 400 microwells cm ² per at least 500 microwells cm ² per at least 750 micro-wells, per cm ² of at least 1,000 microwells per cm ² of at least 2,500 microwells per cm ² of at least 5,000 microwells least 7,500 microwells per cm @ 2, per cm ² of at least 10,000 micro-wells per cm ² of at least 50,000 micro-wells per cm ² It may have a microwell surface density of at least 100,000 microwells or per cm ² of at least 160,000 microwells.

  The microwell size can range from nanoscale (eg, a diameter of about 1 to about 100 nanometers) to microscale or more. For example, each microwell can have a diameter of about 1 μm to about 800 μm, a diameter of about 25 μm to about 500 μm, or a diameter of about 30 μm to about 100 μm. The diameter of the microwell can be about 1 μm or less than 1 μm, about 5 μm or less than 5 μm, about 10 μm or less than 10 μm, about 25 μm or less than 25 μm, about 50 μm or less than 50 μm, about 100 μm or less than 100 μm, about 200 μm or less than 200 μm , Less than about 300 μm or less than 300 μm, less than about 400 μm or less than 400 μm, less than about 500 μm or less than 500 μm, less than about 600 μm or less than 600 μm, less than about 700 μm or less than 700 μm, or less than about 800 μm or less than 800 μm.

  The microwell can have a depth of about 500 μm to about 5000 μm, a depth of about 1 μm to about 500 μm, or a depth of about 25 μm to about 100 μm. The depth that the microwell can have is about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about It can be 1,000 μm, about 1,500 μm, about 2,000 μm, about 3,000 μm, or about 5,000 μm.

  Each microwell may have a circular, hexagonal or square opening. Each microwell can have a sidewall. Their side walls may have a vertical, inclined or curved cross-sectional shape. At least one unique position-specific tag is assigned to at least one microwell of the high density macrowell array to further identify species and to specific microwells of the high density array of microwells, as further described below. The species correlation can be easily achieved. The at least one unique tag can be assigned and / or placed on the bottom and / or at least one side of the one microwell. The at least one specific tag is a target specific nucleotide sequence for annealing to a target nucleic acid fragment of at least one biological entity and a position specificity for identifying at least one microwell of a high density array of microwells Nucleic acid molecules with nucleotide sequences can be incorporated.

  For example, the substrate of a microfabricated device or chip can have a surface that is approximately 4 inches by 4 inches. The surface can define an array of approximately 100 million microwells. The microwell array can be divided into about 100 small compartments by walls and / or the substrate can define an array of about 100 wells, i.e. about 1 million microwells defined within each subcompartment or well total about There will be 100 million microwells. For use cases where different nutrients are tested, microorganisms from natural environmental samples can be loaded onto the chip such that individual microorganisms or microbial clusters are distributed to the microwells on the chip, where each microwell is more Located at the bottom of a large well. Each larger well can incorporate an experimental unit so that 100 different nutrients can be tested in parallel or sequentially on the same chip, ie each well can serve up to 1 million test examples Can do.

  Target cells can be archaea, bacteria or eukaryotes (eg fungi, plants or animals). For example, the target cell may be a microorganism such as an aerobic, anaerobic and / or conditional aerobic microorganism. Different nutrients can be tested in parallel or sequentially using the composition of the target cells, for example to analyze and compare growth or other effects on cell populations, cell components and / or cell products. The composition of the target cell is, for example, one or more viruses (eg bacteriophage), cell surface (eg cell membrane, cell wall), metabolites, vitamins, hormones, neurotransmitters, antibodies, amino acids, enzymes, proteins, sugars, ATP , Lipids, nucleosides, nucleotides, nucleic acids (eg DNA or RNA), phenotypes, mutants, metabolic pathways, genes and adaptability can be examined for cellular components, products, and / or functions.

  The composition of the cells can include natural environment sample extracts and / or dilutions. The natural environment sample extract and / or dilution may contain one or more biological tissues (eg, connective tissue, muscle, nerve, epithelium, plant epidermis, vascular bundle, soil, etc.), biological fluids or other biological products (eg, , Amniotic fluid, bile, blood, cerebrospinal fluid, earwax, exudate, feces, gastric fluid, interstitial fluid, intracellular fluid, lymph fluid, milk, nasal discharge, rumen contents, saliva, sebum, semen, sweat, urine, vaginal secretion , Suspensions, etc.), microbial suspensions, air (eg, with various gas components), supercritical carbon dioxide, soil (eg, with minerals, organics, gases, liquids, microorganisms, etc.), (eg, agriculture) Sediment, living organic matter (eg, plants, insects, other small organisms and microorganisms), dead organic matter, horse grass (eg, pasture, legumes, storage pasture, crop residues, etc.) , Minerals (eg animal, plant, petrochemical Oils or oil products, alcohol, buffers, organic solvents, water (eg natural fresh water, drinking water, sea water, etc.) and / or sewage (eg sanitary, commercial, industrial and / or agricultural wastewater, and surface runoff) Material) and others.

  One method may include combining the cells with a natural environment sample extract and / or dilution prior to applying (eg, loading) the composition comprising the cells to the microfabricated device. . The method may further comprise liquefying the natural environment sample extract and / or dilution. The cell concentration within the composition can be tailored to the target distribution of one cell per experimental unit, well or microwell.

  If the sample contains multiple cells or viruses, the cells in the sample can be lysed after application to the microfabricated device to release the nucleic acid molecules. Cells can be lysed using chemical treatments such as alkaline exposure, detergent, sonication, proteolytic enzyme K or lysozyme exposure. Cells can also be lysed by heating.

  FIG. 5A is an operational step diagram illustrating one method for isolating cells from a sample according to embodiments. In step 500, a sample is obtained. In step 502, the sample is homogenized and / or dispersed using at least one of physical techniques (eg, mixing and / or sonication) and chemical techniques (eg, chelating agents, detergents and / or enzymes). Turn into. In step 504, cells in the homogenized and / or dispersed sample are obtained by density centrifugation using, for example, Nycodenz® non-particulate media (available from Progenbiotechnic GmbH, Heidelberg, Germany). To be separated.

  FIG. 5B is a list diagram illustrating a method for isolating cells from a single soil sample according to embodiments. Panel 506 displays a soil sample. Panel 508 displays the sample homogenized and / or dispersed in the test tube. Panel 510 displays the sample separated into soluble sediment 512, cells 514, insoluble sediment 516 and Nycodenz® 518 after centrifugation.

  FIG. 6 is an operational step diagram illustrating a method for isolating and culturing cells from a sample according to embodiments. In step 600, a sample is obtained. In step 602, at least one cell is extracted from the obtained sample. In step 604, at least one high density microwell array of a microfabricated device or chip is loaded with the at least one extracted cell. Step 604 may incorporate a cell concentration with the at least one extracted cell, select at least one nutrient / medium, and / or incorporate at least one membrane selection. In step 606, at least a portion of the microwell array is sealed with at least one selected membrane to maintain the cell concentration for those microwells. In step 608, the chip is warmed for culture purposes. Step 608 may incorporate temperature selection, determination of atmosphere (eg, aerobic or anaerobic), and / or determination of incubation time. In step 610, the chip is split and / or substantially replicated (eg, using a picker), resulting in two parts of cells cultured according to the methods described herein. For example, at least one membrane may be peeled off with a scissor with a portion of the cultured cells attached, or may be peeled off or otherwise broken to remove the cultured cells as a sample. In optional step 612, a portion of the cultured cells is sacrificed for identification. Step 612 may incorporate PCR, sequencing, and / or various data analysis. In step 614, a strain of the object is identified. Further culture, testing, and / or identification can be performed using, for example, the strain of the subject and / or the remainder of the cultured cells.

  FIG. 7 is a list diagram illustrating a method of isolating and culturing a composite sample according to multiple embodiments. Panel 700 shows an example of a composite sample, particularly a microflora sample 702 and a soil sample 704. In panel 706, at least one cell has been extracted from the sample using, for example, the protocol illustrated in FIGS. 5A and 5B. In panel 708, at least one extracted cell (and any natural environment extract and dilution) is loaded into a microfabricated device or chip having at least one high density microwell array 710. The chip 710 and the reagent cartridge 712 may be inserted into the incubator 714. Reagents can serve to add liquids to maintain nutritional needs for growth and / or various selection purposes. Panel 716 shows the output, i.e., an isolated strain of cultured cells.

  Identify DNA sequencing, nucleic acid hybridization, mass spectrometry, infrared spectroscopy, DNA amplification, genetic factors or other species identifiers to identify the species or taxonomy of cells or microorganisms growing in one microwell Techniques including antibody binding and others to do so are needed. Many identification methods and processing steps kill microorganisms, hindering further culture and study of the subject microorganisms. In order to enable both subsequent cell cultures, research, and further growth of clones of a particular object while allowing cell or microbe identification, location conservation and microbial individuals across several experimental units, wells or microwells in general. Further embodiments are designed to take samples from each experimental unit, well or microwell over a single substrate or chip while maintaining group separation.

  As described above, the substrate may allow a sample to be examined from a cell population using a number of systems, kits, devices and methods. For example, a harvesting device can be applied to the first surface of the substrate. The device may incorporate at least one protrusion that faces the first surface. The at least one protrusion has a diameter smaller than the opening diameter of each microwell, well or experimental unit. The at least one protrusion is a cell population such that a portion of a population of cells in at least one microwell, well or experimental unit attaches and / or binds to at least one protrusion. Can be inserted into at least one microwell, well or experimental unit. A sample of a cell population in at least one microwell, well, or experimental unit has a portion of the cell population in at least one microwell, well, or experimental unit attached and / or bound to at least one protrusion. Can be recovered by removing the harvesting device from the first surface of the substrate. Each protrusion may be a single pin or multiple pins or pin assemblies.

  8A-8C are illustrations of sampling with a single pin or multiple pins in accordance with embodiments. Chip 800 is provided for inspection by microscope 802 and collection by collection controller 804. In FIG. 8A, the sampling control device 804 includes an arm having only one pin 806. In FIG. 8B, an arm having a number of pins 808 is shown. FIG. 8C is a perspective view of the chip during the harvesting step.

  9A to 9D are images actually selecting one well according to a plurality of embodiments. In FIG. 9A, the well is full. In FIG. 9B, the pin has been moved into place. In FIG. 9C, the well is selected. In FIG. 9D, a sample has been removed from the well.

  FIGS. 10A-10D are listings illustrating a tool for collecting from a chip according to embodiments. In FIG. 10A, an instrument with multiple pins is aligned with a chip having multiple wells. In FIG. 10B, the instrument is lowered so that the pin is immersed in the well. In FIG. 10C, pins with attached samples are shown and the samples are transferred to a new chip. On the other hand, in FIG. 10D, the instrument is turned upside down so that the sample is held by the instrument itself.

  FIG. 11 is an image of a well penetrated through a thin agar layer, illustrating a membrane or sealing layer penetration according to embodiments.

  On the other hand, when at least one protrusion is immersed in at least one microwell, well, or experimental unit, a portion of the cell solid group within the at least one microwell, well, or experimental unit has its at least one protrusion The amount moved above and around the object, at least some of which is above the first surface of the substrate and / or the inner surface of the at least one microwell, well or experimental unit To do. The method also includes sampling the cell solid mass within the at least one microwell as a sample by collecting at least some of the transferred portion of the cell solid mass.

  A similar harvesting device can be applied to the second surface facing the first surface of the substrate. The device may incorporate at least one protrusion that faces the second surface. The at least one protrusion has a diameter approximately equal to or smaller than the diameter of the at least one microwell, well or experimental unit. The at least one protrusion is pushed to the second surface at a location corresponding to at least one microwell, well or experimental unit holding a solid group of cells and / or at least one of holding the solid group of cells. Inserted into one microwell, well, or experimental unit, and a portion of the cell population in the at least one microwell, well, or experimental unit is transferred to the first surface of the substrate and / or the at least one micro It is assumed to move above the internal surface of the well, well or experimental unit. The moving part of the cell population can then be collected. The cell population is placed on a plug in at least one experimental unit, well or microwell (eg, other soft material such as hydrogel or agar) and at least one protrusion is pushed to the second surface. And when it is at least one inserted into at least one microwell, the plug is moved, thereby moving part of the cell population.

  A sample of the cell population from at least one experimental unit, well or microwell can be stored in another location. Prior to further sampling, at least one protrusion may be washed and / or sterilized. At least a portion of the at least one protrusion can be composed of a material that has been treated and / or applied with a surface chemical modifier for surface properties that favor cell attachment. The at least one protrusion may be a protrusion array. In applying the device to the first surface of the substrate, the protrusion array can be inserted into a corresponding array of experimental units, wells or microwells. The number of protrusions in the protrusion array can correspond to the number of experimental units in the first array, the number of microwells in one second array of microwells, or the total number of microwells in the substrate.

  Another device for sampling cellular solids within a substrate incorporates at least one needle and / or nanopipette that faces the first surface. The at least one needle and / or nanopipette has an outer diameter that is smaller than the opening diameter of each microwell and an inner diameter that can correspond to the target cell diameter. The at least one needle and / or nanopipette is inserted into at least one experimental unit, well or microwell holding a population of cells. The sample of the cell population in the at least one experimental unit, well or microwell is collected into the device using pressure to pull the cell population out of the at least one experimental unit, well or microwell.

  A sample of the cell population from the at least one experimental unit, well or microwell can be stored in another location. Prior to further sampling, the at least one needle and / or nanopipette may be washed and / or sterilized. The at least one needle and / or nanopipette can be a needle array and / or a nanopipette array. In applying the device to the first surface of the microfabricated substrate, the needle and / or nanopipette array can be inserted into a corresponding array of experimental units, wells or microwells. The number of needles and / or nanopipettes in the needle and / or nanopipette array corresponds to the number of experimental units in the first array, the number of microwells in another one of the microwells or the total number of microwells in the substrate. obtain.

  Another method of sampling a cell population within a substrate includes applying focused acoustic energy to at least one experimental unit, well or microwell holding the cell population within a fluid. . Focused acoustic energy may be applied in an effective way to eject a single droplet from at least one microwell, such as ejection of a small droplet by acoustic waves (ADE) (eg, Sackmann et al., "Acoustical Micro- and Nanofluidics: Synthesis, Assembly and Other Applications," Proceedings of the 4th European Conference on Microfluidics (December 2014)). The small droplet contains a sample of the cell population in at least one experimental unit, well or microwell. The small droplets can be delivered towards another container or surface or substrate.

  The substrate includes at least a first piece that includes at least a portion of the first surface and a second piece that includes at least a portion of the second surface. The first piece and the second piece are detachably connected along at least a portion of a plane parallel to the first surface and the second surface. The plane divides several experimental units, wells or microwells. The cell population in the at least one experimental unit, well or microwell is, for example, a first portion of the cell population in at least one experimental unit, well or microwell attached to the first piece and at least one thereof. Samples are taken by separating the first piece and the second piece while the second part of the cell population in one experimental unit, well or microwell remains attached to the second piece.

  FIG. 12 is a diagram showing a cross section of a chip 1200 according to a plurality of embodiments. Chip 1200 includes a substrate that defines a well array 1202 filled with contents. The base material is composed of a first piece 1206 and a second piece 1208. The first piece 1206 and the second piece 1208 are detachably coupled along a plane parallel to the well array 1202 and bisecting it. When the first piece 1206 and the second piece 1208 are separated, the plurality of wells 1202 and their contents 1204 are split to provide two copies of the contents 1204 that maintain the isolation and position of the contents 1204 of the chip 1200. Bring.

  Each microwell, experimental unit or microchannel is partly separated into a first part and a lower part such that the cell population can grow in both the first part and the lower part. Partial barriers can be included. Prior to sampling the cell population, the method described above may include dispersing and / or reducing the mass within the cell population. Cell mass dispersion and / or reduction within a cell population includes, but is not limited to, sonication, shaking, dispensing with small particles, and the like.

  The above method may further comprise attaching a cell population sample from at least one experimental unit, well or microwell to another location. The other location can be a corresponding experimental unit, well or an array of microwells. The other location may be a single container. A sample of the cell population from at least one experimental unit, well or microwell can be maintained for subsequent culture. On the other hand, cells remaining in the at least one experimental unit, well or microwell in the cell population can also be maintained for the next culture.

  The method may further comprise identifying at least one cell from a sample of the cell solid group and / or a residual cell of the cell population. This can include performing DNA, cDNA and / or RNA amplification, DNA and / or RNA sequencing, nucleic acid molecule hybridization, mass spectroscopy, and / or antibody binding. Alternatively, or in addition, it can also include identifying the experimental unit, well or microwell from which at least one cell was derived. Each experimental unit, well or microwell can be marked with a specific tag that incorporates a position-specific nucleotide sequence. To identify the experimental unit, well or microwell, a position-specific nucleotide sequence is identified in a sequencing and / or amplification reaction, and the position-specific nucleotide sequence is at least the one from which the at least one cell was derived. It can be interrelated with one experimental unit, well or microwell.

  Microfabricated devices such as those described above may allow culturing cells in a sample from the natural environment using additional systems, kits, apparatus and methods. For example, the sample can be applied to the first surface of the substrate such that at least one of the cells occupies at least one microwell, well or experimental unit. A semipermeable membrane is applied to at least a portion of the first surface (eg, at least a portion of the internal surface of the experimental unit or well) so that nutrients can diffuse into the at least one microwell, well or experimental unit. Meanwhile, occupying cells are prevented and / or reduced from leaking out of the at least one microwell, well or experimental unit. The semipermeable membrane can be, for example, a hydrogel layer. The semipermeable membrane is reversibly or irreversibly bound to or pasted on the substrate, for example, by laminating. In this way, occupying cells can be cultured in at least one microwell, well or experimental unit with at least one nutrient. The cells can be gradually transferred from at least one nutrient to at least one other nutrient or nutrient composition using progressive partial exchange over a period of time, thereby undergoing domestication or remodeling.

  The first nutrient derived from the natural environment can be used to culture cells occupying at least one first experimental unit, well or microwell, and the second nutrient derived from the environment is at least It can be used to culture cells occupying one second experimental unit, a well or a microwell. The above method compares the cells occupying at least one first experimental unit, a well or a microwell with the cells occupying at least one second experimental unit, a well or a microwell, and compares the first nutrient and the second nutrient. The step of analyzing may be included.

For example, one method may include one or more of the following steps:
Obtain chips that define 10 to 10 million microwells in many larger wells or flow cells, where each of the microwells has a diameter of about 1 μm to about 800 μm and a depth of about 1 μm to about 800 μm And the chip has one or more surface chemical agents configured to facilitate migration of target microorganisms to the microwells;
Applying a natural environmental sample or a derivative from the natural environmental sample to the chip so that any target microorganism is placed in the microwell;
One or more semi-permeable filters, hydrogel layers on the chip to create a barrier that allows nutrients to diffuse into the microwell but prevent and / or reduce microbial leakage from the microwell Or installing other barriers;
Warming the chip with at least one nutrient (eg from the natural environment) for culture purposes;
Gradually changing the nutrient source by progressive partial exchange using at least one other nutrient (eg, a formulation); and detecting any growth of microorganisms in the microwell.

  Target cells can be archaea, bacteria or eukaryotes. The target virus can be a bacteriophage. If virus is targeted, the microwells of the chip can also contain host cells in which the virus can grow. Occupying cell or virus growth detection may involve detection of changes in biomass (eg, DNA / RNA / protein / lipid), presence of metabolites, pH, nutrient consumption, and / or gas consumption. Occupant cell or virus growth detection includes real-time sequential imaging, microscopic analysis, optical densitometry, fluorescence microscopic analysis, mass spectrometry, electrochemistry, amplification (DNA, cDNA and / or RNA), sequencing (DNA and / or Or RNA), nucleic acid molecule hybridization, and / or antibody binding.

  FIG. 13 is a work step diagram illustrating a method for performing selection according to multiple embodiments. In step 1300, a sample is obtained. In step 1302, at least one cell is extracted from the acquired sample. In step 1304, at least one high density microwell array of microfabricated devices or chips is loaded with the at least one extracted cell. Step 1304 may include providing a cell concentration having the at least one extracted cell, selecting at least one nutrient / medium, and / or selecting at least one membrane. In step 1306, at least a portion of the microwell array is sealed with at least one selected membrane to maintain the cell concentration for those microwells. In step 1308, the chip is warmed for culture purposes. Step 1308 may include temperature selection, determination of atmosphere (eg, aerobic or anaerobic), and / or determination of incubation time. Genetic testing and / or functional testing may be performed. In step 1310, genetic testing is performed on the chip. In step 1312, the chip is split and / or substantially replicated (eg, using a picker) to yield two parts of cells cultured according to the methods described herein. For example, the at least one membrane may be peeled off, peeled off or pierced so that a portion of the cultured cells remain attached to sample the cultured cells. In optional step 1314, a portion of the cultured cells is sacrificed for identification. Step 1314 may incorporate PCR, sequencing, and / or various data analysis. In step 1316, the strain of the object is identified. Further culture, testing, and / or identification can be performed using, for example, the strain of the subject and / or the remainder of the cultured cells. On the other hand, in step 1318, a function test is performed on the chip. In step 1320, one or more variables are observed and the strain of interest is identified, as in step 1316.

  FIG. 14 is a list diagram illustrating a screening method according to a plurality of embodiments. Panel 1400 shows an example of a composite sample, specifically a microbial community sample 1402 and a soil sample 1404. In panel 1406, at least one cell has been extracted from the sample using, for example, the protocol shown in FIGS. 5A and 5B. In panel 1408, at least one extracted cell (and any environmental extract and / or dilution) is loaded into a microfabricated device or chip with at least one high density microwell array 1410. Chip 1410 and reagent cartridge 1412 may be inserted into incubator 1414. Reagents can be useful for adding liquids to maintain nutritional requirements for growth and / or various sorting purposes. Panel 1416 shows the output, ie the sorting result and the isolated strain of cultured cells.

  FIG. 15 is a series of images showing a selection example according to a plurality of embodiments. The images show multiple portions of the chip with a membrane and acid sensitive layer applied to test for low pH. In the image 1500, more than 1800 50 μm microwells are displayed with nine distinct hits 1502. Image 1504 is a magnified screen of enclosure 1504 and image 1506 is a magnified screen of one of the microwells having hit 1502.

  FIGS. 16A-16C are images illustrating collection from an obstruction according to multiple embodiments. In FIG. 16A, at least one well has been drilled using a collector with a microscope and at least one pin. In FIG. 16B, the pins are removed and cultured in medium. In FIG. 16C, the proliferation is apparent.

  FIG. 17A is an exploded view illustrating a sorting chip according to a plurality of embodiments. In FIG. 17A, chip 1700 incorporates a high density array of microwells with, for example, soil microorganisms inside. A film 1702 is attached to the chip 1700. A gasket 1704 is attached to the chip 1700 through the membrane 1702. Agar 1706 with fluorescent E. coli is attached to chip 1700 through gasket 1704 and membrane 1702. FIG. 17B and FIG. 17C are images for explaining a selection example according to a plurality of embodiments. In this example, the shield is for the exclusion area. FIG. 17B is a fluorescence image of a chip processed like the chip 1700 in FIG. 17A and accompanied by a shield. FIG. 17C is an image showing the steps of collecting a sample from this chip through agar.

  In embodiments, after a portion of a sample (or a portion of that portion) has been removed from the device, the location on the device can be correlated with that portion of the sample present at that location. The device may be a microarray or may incorporate a microarray. The microarray has multiple locations where the sample is applied, where after a portion of the sample is removed from the microarray, each location is marked with a unique tag that can be used to identify the location from which that portion of the sample originated. It is attached.

  The present invention relates to a method for identifying where in a microarray a part of a sample containing at least one nucleic acid molecule has been derived after a part of the sample has been removed from the microarray, the method comprising (a) Applying one or more portions of the sample to one or more of a plurality of positions on the microarray, wherein each position comprises (i) a position-specific nucleotide sequence and (ii) a first target-specific nucleotide (B) annealing the target nucleic acid molecule found in at least a portion of the sample to the position marking tag, wherein the marking is marked with a specific tag having a nucleic acid molecule comprising a sequence; The target nucleic acid molecule population is subjected to primer extension, reverse transcription, single-stranded ligation reaction or double-stranded ligation reaction, thereby position-specific nucleotide sequence Incorporating into each nucleic acid molecule generated by primer extension, reverse transcription, single-stranded ligation or double-stranded ligation, (d) combining the nucleic acid molecule populations produced in step (c), (e) Sequencing the combined nucleic acid molecule population, thereby obtaining that sequence among one or more position-specific nucleotide sequences, and (f) at least one position obtained from the combined nucleic acid molecule population Associating that sequence of specific nucleotide sequences with a location on a microarray marked with a tag comprising the location specific nucleotide sequence, thereby identifying the location on the microarray from which a portion of the sample containing at least one nucleic acid molecule was derived. Identifying. In embodiments, the sample can include at least one cell from which one or more nucleic acid molecules are released after step (a) and before step (b). The sample can contain at least one cell, and the at least one cell self-replicates or divides after step (a) and before step (b). A portion of the portion of the sample can be removed from at least one location prior to step (b) so that the portion of the portion of the sample is the original location of the portion of the sample on the microarray. Can be stored in correlated containers. The method of correlating or identifying positions may further comprise amplifying the population of nucleic acid molecules produced in step (c) or the combined nucleic acid molecules produced in step (d). The amplification steps include polymerase chain reaction amplification, multiplex polymerase chain reaction amplification, nested polymerase chain reaction amplification, ligase chain reaction amplification, ligase detection reaction amplification, strand displacement amplification, transcription-based amplification, and nucleic acid sequence-based amplification. It may include amplification, rolling circle amplification, or hyperbranched rolling circle amplification. Additional primers can be added during the amplification reaction. For example, both 5 'and 3' primers may be required for PCR reactions. One of the primers used during the amplification reaction can be complementary to the nucleotide sequence in the sample.

  In embodiments, a composition comprising cells and / or viruses is treated with a nucleolytic enzyme before the composition is applied to a microfabricated device so that contaminating nucleic acid molecules are not amplified in subsequent steps. Can do.

Sequencing used in the methods and apparatus of the present invention may be any process that obtains sequence information including hybrid formation and the use of sequence specific proteins (eg, enzymes). Its sequencing includes Sanger sequencing, sequencing by hybrid formation, sequencing by ligation, quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer, molecular labeling, TaqMan reporter probe Digestion, pyrosequencing, fluorescence in situ sequencing (FISSEQ), fluctuation sequencing, multiple sequencing, polymerized colony (POLONY) sequencing (see, eg, US Patent Application Publication No. 2012/0270740, which is herein incorporated by reference) Incorporated herein by reference in its entirety), nanogrid rolling circle (ROLONY) sequencing (see, eg, US Patent Application Publication No. 2009/0018024, which is incorporated herein by reference in its entirety) Incorporated in the description of the invention), allele-specific oligo-ligation analysis or sequencing, or Cash Sequencing (NGS) may include sequencing by the platform. Non-limiting examples of NGS platforms include Illumina® (San Diego, California) (eg, MiSeq ^™ , NextSeq ^™ , HiSeq ^™ and HiSeqX ^™ ), Life Technologies (Carlsbad, California) (eg, Ion Torrent ^™ ) and Pacific. A system of Biosciences (Menlo Park, Calif.) (Eg, PacBio® RS II) is included.

  An organism or species can be identified by comparing the nucleic acid sequence obtained from that organism with various databases that contain sequences of numerous organisms. For example, ribosomal RNA sequence data is available from the SIL VA rRNA database project (Max Planck Institute for Marine Microbiology, Bremen, Germany (www.arb-silva.de)), eg Quast et al., “The SIL VA Ribosomal RNA Gene Database Project: Improved Data Processing and Web-Based Tools, "41 Nucl. Acids Res. D590-D596 (2013), and Pruesse et al.," SINA: Accurate High-Throughput Multiple Sequence Alignment of Ribosomal RNA Genes, "28 Bioinformatics 1823 -1829 (2012), both of which are incorporated herein by reference in their entirety. Other ribosomal RNA sequence databases are available from the Ribosome Database Project (Michigan State University, East Lansing, Michigan (www.rdp.cme.msu.edu): eg Cole et al., “Ribosomal Database Project: Data and Tools for High Throughput rRNA Analysis 42 Nucl. Acids Res. D633-D642 (2014). This article is hereby incorporated by reference in its entirety) and Greengenes (Lawrence Berkeley National Laboratory, Berkeley, California). (www.greengenes.lbl.gov): See, for example, DeSantis et al., “Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB,” 72 Appl. Environ. Microbial. 5069-72 (2006). This article includes the entire text of the present specification, which is incorporated herein by reference. The GenBank® gene sequence database is a publicly available nucleotide sequence of nearly 260,000 officially described species (National Institutes of Health, Bethesda, Maryland (www.ncbi.nlm.nih.gov); For example, Benson et al., “GenBank,” 41 Nucl. Acids Res. D36-42 (2013)).

  The verification and identification sequence may include a 16S ribosomal region, an 18S ribosomal region, or some other region that provides identification information. The desired variant can be a species that includes a genotype (eg, a single nucleotide polymorphism (SNP) or other type of variant) or a specific gene sequence (eg, a sequence that specifies the genetic code of an enzyme or protein). An organism or species can also be identified by checking its sequence against a national custom sequence database. In some cases, if the sequence obtained from a portion of the sample at a certain position is at least a predetermined percentage of identity to a known DNA, cDNA or RNA sequence obtained from a species or microorganism (eg, at least 90%, At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) It can be concluded that the species or organism is found at that location on the microarray.

  The invention further relates to a method of manufacturing a microarray having a plurality of positions for applying a sample, wherein at least one position is marked with a unique tag. The method comprises the steps of: (a) synthesizing a plurality of tags each comprising (i) a position-specific nucleotide sequence and (ii) a nucleic acid molecule comprising a target-specific nucleotide sequence; and (b) a plurality of tags on the microarray. Placing the tag at at least one of the positions. In another embodiment, the invention relates to a method of manufacturing a microarray having a plurality of locations for applying a sample, at least one of which is marked with a unique tag, The method comprises the steps of: (a) synthesizing a plurality of tags each comprising a nucleic acid molecule comprising a target specific nucleotide sequence and no position specific nucleotide sequence; and (b) in a plurality of positions on the microarray. Placing the tag in at least one position. The target specific sequence can be the same at any location in the microarray. In any of the above embodiments, step (a) may be performed before step (b). Placement step (b) includes placing the tag at each position by a liquid handling procedure (eg, pipetting, spotting using a solid pin, spotting using a hollow pin or deposition by an inkjet device). The at least one tag can comprise a nucleic acid molecule or a portion of a nucleic acid molecule prior to synthesis. Step (a) may be performed simultaneously with step (b). In some embodiments, the at least one tag includes a nucleic acid molecule that is synthesized at each position by in situ synthesis. The synthesis step (a) can include ink jet printing synthesis or photolithography synthesis.

  Each location of the microarray can be configured to receive a portion of the sample. The position may be tagged or labeled with at least one (i) a position-specific nucleotide sequence (eg, a barcode) and (ii) a nucleic acid molecule (eg, an oligonucleotide) containing a target-specific nucleotide sequence. I can do it. The target specific nucleotide sequence can complement or generally complement the nucleotide sequence found in the sample. The order of the nucleotide sequence from the 5 'end to the 3' end of the nucleic acid molecule may be (1) a position-specific nucleotide sequence, (2) a target-specific nucleotide sequence. On the other hand, the order of the nucleotide sequence from the 5 'end to the 3' end of the nucleic acid molecule may be (2) then (1). The nucleic acid molecule can be attached to the microarray at its 5 'end. One or more locations of the device (eg, microarray) may be untagged or unlabeled.

  The term “complementary” or “generally complementary” means between nucleotides or between nucleic acids, eg, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid. Can mean hybrid formation, base pairing or duplex formation. Complementary nucleotides are generally A and T / U or C and G. A single stranded nucleotide that is optimally aligned, compared and with appropriate nucleotide insertions or deletions is at least about 80%, usually at least about 90% to 95%, more preferably about When paired with 98% to 100%, two single stranded RNA or DNA molecules are said to be substantially complementary. On the other hand, substantial complementarity exists when an RNA or DNA strand is hybridized to its complement under selective hybrid formation conditions. Typically, selective hybrid formation occurs when there is at least about 65%, at least about 75%, or at least about 90% complementarity over a stretch of at least 14 to 25 nucleotides.

  The terms “selectively form hybrids” or “selective hybrids” can mean to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof are selectively hybridized to nucleic acid strands under hybridization and washing conditions that minimize a significant amount of detectable binding to non-specific nucleic acids. “Highly stringent” or “highly stringent” conditions can be used to achieve selective hybrid formation conditions known in the art and described herein. An example of “highly stringent” or “highly stringent” conditions is a method of culturing one polynucleotide with another polynucleotide, in which one polynucleotide is 6 × SSPE or SSC, 50% formaldehyde. 5x Denhardt's reagent, 0.5% SDS, 100 μg / ml denatured fragmented sperm DNA in a hybrid formation buffer and attached to a membrane-like solid surface for 12-16 hours at a hybrid formation temperature of 42 ° C. Then, it is washed twice at 55 ° C. using a washing buffer of 1 × SSC and 0.5% SDS.

  The nucleic acid molecule that is part of the position tag can comprise at least one deoxyribonucleotide or at least one ribonucleotide. The nucleic acid molecule may be single-stranded or double-stranded. The nucleic acid molecule may be double stranded with a single stranded overhang.

  In embodiments, position tags can be used to amplify nucleic acid molecules that anneal. Thus, the position tag can comprise a nucleic acid sequence further having an amplification primer binding site. The amplification primer binding site is at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least It may be 30 nucleotides in length. The order of the nucleotide sequence from the 5 ′ end to the 3 ′ end of the nucleic acid molecule can be, for example, (1) an amplification primer binding site, (2) a position-specific nucleotide sequence, and (3) a target-specific nucleotide sequence. .

  In embodiments, the nucleic acid molecule can comprise a target-specific nucleotide sequence without comprising a position-specific nucleotide sequence. In certain embodiments, the nucleic acid molecule can comprise a target-specific nucleotide sequence without comprising either a position-specific nucleotide sequence or an amplified binding site sequence. In further embodiments, the nucleic acid molecule may comprise only target-specific nucleotide sequences. In still further embodiments, the nucleic acid molecule can be composed solely of target-specific nucleotide sequences. Amplification primer binding sites are based on polymerase chain reaction primer, multiple polymerase chain reaction primer, nested polymerase chain reaction primer, ligase chain reaction primer, ligase detection reaction primer, strand displacement primer, transcription-based primer, nucleic acid sequence It may be capable of binding to a primer, rolling circle primer, or hyperbranched rolling circle primer. Additional primers can be added to the microarray during the amplification reaction. For example, both 5 'and 3' primers may be required for PCR reactions. The target-specific nucleotide sequence is amplified at the location of the target nucleic acid molecule and can be detected by, for example, qPCR, endpoint PCR and / or dye to detect the amplified nucleic acid molecule.

It may be desirable to read the sequence of such nucleic acid molecules that anneal to the amplified product based on the position tag or nucleic acid molecule. The position tag can include a nucleic acid sequence that further includes an adapter nucleotide sequence. In certain embodiments, the adapter nucleotide sequence may not be found within the position tag, but is added to the sample nucleic acid molecule in a secondary PCR reaction or by a ligation reaction. The adapter nucleotide sequence can be a universal adapter or an adapter for a specific sequencing platform (eg, Illumina® or Ion Torrent ^™ ). The adapter nucleotide sequence can include a sequencing primer binding site. Sequencing primer binding sites include Sanger sequencing, sequencing by hybrid formation, sequencing by ligation, quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer, molecular labeling, TaqMan Reporter probe digestion, pyrosequencing, fluorescence in situ sequencing (FISSEQ), fluctuation sequencing, multiple sequencing, polymerized colony (POLONY) sequencing (see, eg, US Patent No. 2012/0270740), nanogrid rolling circle ( ROLONY) can bind primers for sequencing (see, eg, US Patent No. 2009/0018024), allele-specific oligo-ligation analysis, sequencing on the NGS platform, or any suitable sequencing procedure obtain. Non-limiting examples of NGS platforms include Illumina® (San Diego, California) (eg, MiSeq ^™ , NextSeq ^™ , HiSeq ^™ and HiSeqX ^™ ), Life Technologies (Carlsbad, California) (eg, Ion Torrent ™) and Pacific Biosciences Systems from (Menlo Park, California) (eg PacBio® RS II) are included.

  The position-specific nucleotide sequence (eg, barcode) is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 The nucleotide length may be

  The target specific nucleotide sequence is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least It may be 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides in length.

  At least one location on the microarray can be further marked with a unique molecular identifier tag. The unique molecular identifier tag can be used to quantify growth (eg, microbial colony growth or cell replication at that location). The unique molecular identifier can be a disordered nucleotide sequence. Specific molecular identifier usage methods and specific molecular identifier examples have been described in the art, for example see WO 2013/173394, which is hereby incorporated in its entirety by reference. For example, a unique molecular identifier tag can have the nucleotide sequence NNNANNNCNNNTNNNGNNNANNNCNNN (SEQ ID NO: 1), where each amplified molecule has a unique (specific) DNA sequence barcode (4 ^ N barcode) Or N in this example (4 ^ 21-4 trillion), so many Ns (the same random mixture of ACGTs) have created a large coding space. This array can be enumerated without interference from amplification bias or other problems. The fixed bases (A, C, G, T) of SED ID NO: 1 help correct reading of the barcode, for example, handling of insertion deletions.

  The present invention contemplates the use of position specific tags for observing and recording the presence or amount of multiple target specific nucleotide sequences (eg, multiplexing) within a sample. At least one position on the microarray is further marked, for example, with a second specific tag having a nucleic acid molecule comprising (i) an amplification primer binding site, (ii) a position specific nucleotide sequence, and (iii) a target specific nucleotide sequence. Can be attached.

  In embodiments, the nucleic acid molecule can comprise a target-specific nucleotide sequence without comprising a position-specific nucleotide sequence. In certain embodiments, a nucleic acid molecule can comprise a target-specific nucleotide sequence without including either a position-specific nucleotide sequence or an amplified binding site sequence. In further embodiments, the nucleic acid molecule may comprise only target-specific nucleotide sequences. In still further embodiments, the nucleic acid molecule can be composed solely of target-specific nucleotide sequences. Such target specific nucleotide sequences are at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least It may be 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides in length. In certain embodiments, the target-specific sequence can be the same everywhere in the microarray. Additional target specific nucleotide sequences can be observed and recorded. For example, one or more positions can be marked with at least 10, at least 25, at least 50, at least 75, or at least 100 unique tags, where each tag is another target in the tag at that position. It contains one target-specific nucleotide sequence that is different from the specific nucleotide sequence.

  Any locus of interest can provide a target-specific nucleotide sequence. For example, bacterial 16S ribosomal RNA (rRNA), 18S ribosomal RNA, poly (A) RNA, RNA polymerase gene, DNA polymerase gene, RecA gene, transposase gene sequence, ribosome internal transcription spacer region (ITS) sequence, enzyme code Any part of the gene, regulatory region DNA sequences, binding site DNA sequences, or any of these sequences can serve as a target-specific nucleotide sequence. The disclosed system, kit, apparatus or method is described in Sundquist et al., “Bacterial Flora-Typing with Targeted, Chip-Based Pyrosequencing,” 7: 108 BMC Microbiology (2007) and Wang et al., “Conservative Fragments in Bacterial l6S rRNA Genes and Primer. One or more of the bacterial 16S rRNA primers described in Design for l6S Ribosomal DNA Amplicons in Metagenomic Studies, "4: 10PLoS ONE e7401 (2009) may be used. Each of these documents is hereby incorporated in its entirety by reference herein.

  Samples used in the disclosed apparatus and methods can include a plurality of nucleic acid molecules. The sample can contain at least one DNA molecule or at least one RNA molecule. The sample can include at least one nucleic acid molecule formed by restriction enzyme digestion. The sample can include at least one cell (eg, archaeal cell, eubacteria cell, fungal cell, plant cell, and / or animal cell). The sample can include at least one microorganism. The sample may contain one or more viruses (eg, bacteriophages) that may need to be supplied with host cells. The portion of the sample at one location of the microarray may be a single cell or a colony in which a single cell has grown. For example, microorganisms or cells can be individually placed in a microwell, and each individual microorganism or cell can be divided or replicated so that colonies grow inside each microwell in which each microorganism or cell is placed. Can be. Thus, one location of the microarray can accommodate a single microbial species or a mixed community of microbial varieties that help each other grow. The sample can contain any suitable dilution. Samples may include, but are not limited to, soil, sewage, feces, body cavity contents, biological fluid, living organic matter, dead organic matter, microbial suspension, natural fresh water, drinking water, seawater, wastewater, air , Supercritical carbon dioxide, minerals, gases, buffers, alcohols, organic solvents, and / or oils. In embodiments, (a) (i) one position-specific nucleotide sequence and (ii) one or more target-specific nucleotide sequences, or (b) one or more target-specific nucleotide sequences (ie, positions Specific nucleotide sequence) is placed in at least one location of the microarray before a portion of the sample is placed in that location. In other embodiments, (a) (i) a position specific nucleotide sequence and (ii) one or more target specific nucleotide sequences, or (b) one or more target specific nucleotide sequences (ie, position specific Is not placed in at least one position of the microarray after a portion of the sample has been placed in that position. In one example, a sample or a portion of a sample is placed on a microarray with (i) at least one of one position-specific nucleotide sequence and (ii) one or more target-specific nucleotide sequences. It can be placed and cultured before the containing nucleic acid molecule is placed. In embodiments, a portion of the portion of the sample is removed from at least one location of the microarray and stored in another container, or the nucleic acid molecules are placed in at least one location of the microarray. The microarray may be divided either before or after being placed.

  The at least one position-specific tag can comprise a nucleic acid molecule or a portion thereof that has been pre-synthesized and placed at that position by a liquid processing procedure. For example, the liquid processing procedure can be pipetting, spotting using a solid pin, spotting using a hollow pin, or deposition by an inkjet device. The tag can be generated at that location using a number of previously synthesized nucleic acid molecules. The at least one tag can include a nucleic acid molecule that is synthesized at that location by in situ synthesis (eg, ink jet printing or photolithography).

(Digital enumeration of species)
High density chip devices with surfaces having high density microwells are described herein. Microorganisms in the microbiota sample are diluted and applied to the device so that the wells contain approximately one microorganism per occupied well. The chip may then be warmed for culture purposes so that the microorganisms are replicated in the wells. In addition, a DNA location indexing system is described here to determine what species are present in each well. This indexing system provides a number of PCR primers pre-loaded into each well, including addressing barcodes that identify the wells as well as specific genes within the microbial genome that provide species information or target the desired gene sequence It may involve having a primer sequence directed to the element (eg 16S). After incubation, microbial DNA is released, their PCR primers amplify the target bacterial DNA region, and amplification products from various wells on the chip are pooled and can be further decoded with next generation sequencing.

  The systems, kits, devices and methodologies described herein can be used to perform an absolute count of the number of each microbial species or variant within a sample. Each well may represent a digital event that represents the presence of a single microorganism in the original diluted sample. The location indexing system may allow the user to determine what bacterial species are in the well. One building block of measurement may be “a certain bacterial species is in one well” and may be independent of the number of bacteria in that well.

  In one example, a mixed sample of microorganisms includes 50% species 1, 30% species 2, and 20% species 3. The sample is diluted and then each occupied well is applied to the chip so that it has, for the most part, one microorganism. The microorganism is replicated. It should be noted that the replication rate can be different for different species. The chip is then processed so that DNA from the in-well microorganism is released and 16S or some other target sequence is amplified. The DNA amplification product from each well can be pooled and sequenced using next generation sequencing methods. Next generation sequencing data can be analyzed to determine for each well which species is in each occupied well. Many wells may not be occupied at all. The abundance of each species can be determined by dividing the total number of wells occupied by each species by the total number of occupied wells. An absolute abundance determination can be made by multiplying the% abundance of each species from the step by the total number of microorganisms in the original sample. Sequencing data can be compared to publicly available sequencing databases to determine what species are in each occupied well. For example, ribosomal RNA sequences can be obtained from the SIL VA rRNA database project described above. Other ribosomal RNA sequence databases include the Ribosomal Database Project, Greengenes, and GenBank (registered trademark) gene sequence database described above.

Current methods of estimating microbial species abundance in a sample involve the use of traditional techniques such as microscopy, staining, selective media, metabolic / physiological shields and culture using Petri dishes. These methods are often inaccurate due to lack of specificity (microscopy, staining, metabolic / physiological shield) or lack of ability to reveal all species in the sample (selective media, culture) So many species do not grow well or grow at all in traditional methods.
Current molecular methods to determine the relative abundance of microbial species within a microbiota sample extract microbial DNA from the sample, perform PCR amplification of 16S or other DNA regions that provide species or other information, and then The resulting PCR product involves performing next generation sequencing (NGS). The relative abundance of each species in the original sample is inferred from the relative frequency of species-specific DNA sequences in the NGS data. There are numerous references to this type of analysis, and this method supports the study of many microbiota.

  The problem with current methodologies is that the different numbers of 16S genes that can exist in different microorganisms, PCR biases where sequences from different microbial species are amplified at different rates, and sequences from different microbial species differed There is no adjustment to the sequencing bias that can be arranged at a rate. The result is that there are many uncertainties regarding the accuracy of the relative abundance data derived using current methodologies.

  Aggregation of different species can be based on the presence of species in a single well. This is directly related to a single microorganism from the original sample that divides into its wells during loading. Only PCR / NGS can be used to identify what microbial species are present in each well. The number of sequences identified does not form part of the calculation. It is therefore not important whether the method has PCR, NGS or target sequence copy number variation or bias.

Embodiments can be applied to microbiota research, microbial product discovery and development, medical diagnostics, and any other field where accurate counting of microbial species within a sample is desired.
Thus, embodiments provide an even more accurate measurement of the relative abundance of each species in the flora sample, and this relative abundance measurement (to account for dilution and / or microbial activity in the original sample). It may provide the ability to convert to absolute abundance or direct aggregation of each species in the original sample (in combination with total count measurements). Embodiments may provide new applications of microfabricated high density chips (outside of microbial culture and selection).

  FIG. 18 is a work step diagram illustrating a tabulation method according to a plurality of embodiments. In step 1800, a sample is obtained. In step 1802, at least one cell is extracted from the acquired sample. In step 1804, at least one extracted cell is loaded into at least one high density microwell array of a microfabricated device or chip. Step 1804 includes providing a cell concentration with the at least one extracted cell, selecting at least one nutrient / medium, and / or selecting at least one membrane. In step 1806, at least a portion of the microarray is sealed with at least one selected membrane to maintain the cell concentration with respect to the microwell. In step 1808, the chip is warmed for culture purposes. Step 1808 includes selecting the temperature, determining the atmosphere (eg, aerobic or anaerobic), and / or adjusting the incubation time. In step 1810, the cultured cells may be sacrificed for identification. Step 1810 includes PCR, sequencing, and / or various data analysis. In step 1812, information about the sample (eg, microbial community structure) may be evaluated and / or determined.

  FIG. 19 is a list diagram illustrating a tabulation method according to a plurality of embodiments. Panel 1900 shows an example of a composite sample, specifically a microflora sample 1902 and a soil sample 1904. In panel 1906, at least one cell has been extracted from the composite sample using, for example, the protocol shown in FIGS. 5A and 5B. In panel 1908, the at least one extracted cell (and any environmental extract and / or dilution) is loaded into a microfabricated device or chip comprising at least one high density microwell array 1910. Chip 1910 and reagent cartridge 1912 can be inserted into incubator 1914. The reagent may be useful for liquid replenishment and / or various sorting purposes to maintain the nutrients necessary for growth. Panel 1916 shows the output, ie the sequence and the relative abundance of the cultured cells.

(Platform using small droplets)
A platform with isolated small droplets can be used for separation, culture, and / or sorting in much the same way that chips are used. Small droplets are analogs of microwells that function as nano or picoliter containers. The small droplet generation method can be used to separate microorganisms from complex environmental samples, especially when combined with an on-chip cell classifier type instrumentation. Small droplet addition can be utilized to supply microorganisms. Small droplet splitting can be utilized for sequencing or some other degradation test while leaving behind a biological sample. All the preliminary work required for sequencing can be done in the same way in a small droplet system.
Embodiments can be utilized to remove microorganisms from a complex environment into small droplets. For example, a modular system for generating droplets that includes a cell suspension can include one or a few cells. Aqueous droplets can be suspended in an immiscible liquid so that they are not close to each other and do not touch or soil any surface. Small droplets can be produced in each microwell, for example at 30 Hz, which in other words would be millions every day.

  A droplet-based microfluidic system can encapsulate, manipulate, and / or culture small droplets (eg, about 30 pL). It is noted that cell survival and propagation is similar to large volume solution control experiments. Small droplets are generated at hundreds of Hz, which means that millions of droplets can be generated in hours. Single chip based devices can be used to generate small droplets, and the small droplets can be designed to contain single cells.

  Several embodiments can be utilized to sort cells within small droplets. Fluorescence sorting after small droplet culture can be performed on the chip, for example, at a rate of 500 droplets per second. Small droplets can be flowed through one channel at the focal point of an epifluorescence microscope configured for many different measurements. This can be a particularly effective method of sorting for metabolites because the local concentration is quite high because it is trapped in very small droplets.

  Several embodiments can be utilized to classify small droplets. Once the cells are isolated, grown and / or sorted, they can be sorted so that a useful sample is removed. Small droplets can be classified in a manner similar to commonly used FACS mechanisms.

  Embodiments can be utilized to break up small droplets. Embodiments may require the ability to divide the sample in order to obtain the sample and send one part to sequencing (disruption step) and retain another part that is viable medium . There are many ways to break up small droplets, for example building a T-junction with a carefully calculated size that will break as the droplet continues to flow, or electrowetting (applying too high a voltage) However, it is not limited to this.

  Embodiments can be utilized to coalesce droplets and / or add reagents to the droplets. For example, long-term (eg, several weeks) culture of cells requires the ability to add fluid to maintain the nutrients necessary for growth. It may also be advantageous to be able to add reagents for various sorting purposes. Small droplet sorting relies on the ability to combine small droplets containing compound codes with small droplets containing single cells. The small droplets can then be cultured and / or returned to the analysis chip to identify compounds via their codes. This may require the ability to accurately coalesce small droplets as needed.

  Multiple embodiments can be utilized to perform PCR in small droplets. PCR can be used to ultimately sequence specific genetic elements (eg, the 16S region) to identify microorganisms. This can be used to determine what type of microorganism is growing in each well. In systems using small droplets, this technique is used to determine what microorganisms are present in each droplet as long as the appropriate primer sequences are designed to amplify the correct region of the gene. Can be.

  Embodiments can be utilized to sequence DNA from small droplets (eg, generated in a PCR step) and / or create a DNA library.

(Position specific tag for high-density chip)
A high density chip device having a surface with high density microwells can be used. Microorganisms from the microbiota sample can be diluted and applied to the device such that the well contains approximately one microorganism per occupied well. The chip can be warmed for culturing purposes so that the microorganisms replicate in the wells and the resulting solid population represents a single species. A DNA location indexing system can be used to determine what species are present in each well. This indexing system is targeted to pre-loaded PCR primers that contain addressing barcodes that identify the wells and specific genetic elements that provide species information (eg 16S of microbial genes) It may be necessary to have a primer sequence. Microbial DNA can be released after incubation, PCR primers can amplify the target bacterial DNA region, and amplification products from various wells on the chip can be pooled and then decoded by next generation sequencing.

  The above position indexing system may involve incorporating a different position code for each well on the microchip, in other words, so that the total number of codes required to encode a given number of wells is reduced. A composite location code can be incorporated into each well. For example, if there are 100 wells on one chip, 100 codes are required if there is one code per well. If two codes were decoded from each well, the same chip could be coded with only 20 codes (ie, 10 codes for the grid x-axis and 10 codes for the y-axis). .

An example of a PCR strategy that incorporates 2 codes per well is shown in Table 2.

An example of a PCR strategy that incorporates 3 codes per well is shown in Table 3.

  Three oligo primers are used to make a single PCR product. The advantage of this system using two oligos to attach a multi-part barcode to one end of the molecule is, for example, to reduce the maximum length of oligos required or to create exceptionally long barcodes , May be included.

  This approach can be generalized to incorporate n barcodes per reaction. The approach can also include various implementations where there are several barcodes on the same side of the target sequence region. With the addition of NGS sequencing adapters, the entire sequence for a population of barcoded PCR products can be decoded by next generation sequencing.

In another implementation, a fixed code can be added to identify the sample number or plate number, and storage of the composite sample / plate in one stroke toward the two barcodes as shown in Table 4. Can be possible.

全ての場合において配列内のコードの位置が情報を伝えているので、従ってコード１、コード２およびコード３のバーコードは必ずしも個々のウェル内で互いに異なっている必要はない。 In a further implementation situation, a fixed code can be added to identify the sample number or plate number and allows for the storage of composite samples / plates in one step towards the three bar codes as shown in Table 5 Can be.

Since in all cases the position of the code within the sequence conveys information, the code 1, code 2 and code 3 barcodes need not necessarily be different from one another in the individual wells.

  Making a number of oligos and printing a number of chips using a single code encoding system is expensive. For example, a 10,000 well chip requires 10,000 independent barcodes and 10,000 independent prints to place the barcode in the well on the chip. When using a two-code system, only 200 bar codes are required in some cases if printing is repeated 200 times to produce the chips. This means significant savings in oligo costs, printing time and printing capital investment.

  It is highly useful to perform amplification and sequencing analysis after the use of dual barcoded PCR primers to obtain positional data for DNA or RNA containing randomly divided portions on a microfabricated chip. Can have a relatively low cost.

  FIG. 20 is a list diagram illustrating an index creation system according to a plurality of embodiments. Microwell 2000 has N rows and M columns, thereby generating N × M unique indicators. The position of the microwell in the chip 2000 can be considered to have coordinates (N, M). Each column has a common reverse primer sequence (eg, R1, R2, R3,... RM), and each row has a common forward primer sequence (eg, F1, F2, F3,... FN) ). For example, a specific tag directed to a particular genetic element within 16S ribosomal ribonucleic acid (rRNA) can include a forward primer sequence F515 as well as a reverse primer sequence R806. Following chip 2000 PCR, the presence of the target gene element can be mapped back to the original unique microwell based on the presence of the forward and reverse primer sequences. For example, the presence of F515 and R806 guides the user to a microwell having coordinates (515, 806) within chip 2000.

(Reduced variability for PCR amplification products across bacteria-containing microwells)
A DNA location indexing system can be used to determine what species are present in each well. This indexing system targets pre-loaded PCR primers that contain addressing barcodes that identify the wells and specific genetic elements (eg, 16S) within the microbial gene that provide species information. It may be necessary to have a primer sequence that Microbial DNA is released after incubation, PCR primers amplify the target bacterial DNA region, and amplification products from various wells on the chip are pooled and can be decoded by next generation sequencing.

  Embodiments to limit the variation in the amount of PCR product across the well include a chip so that the amount of PCR primer is limiting in the DNA amplification reaction for the majority of the expected sample DNA concentration. Limitations of the amount of PCR primer in the well can be incorporated during the production of the PCR, and thus the amount of PCR product produced will not be as variable throughout the well.

  Embodiments for limiting variation in the amount of PCR product across a well include a chip so that the amount of PCR product produced across the well is less variable compared to a full cycle PCR amplification protocol. Limiting the number of PCR cycles above to less than 3, alternatively less than 5, alternatively less than 10 cycles, alternatively less than 15 cycles, alternatively less than 20 cycles, alternatively less than 25 cycles, alternatively less than 30 cycles.

  Embodiments for limiting the variation in the amount of PCR product across the well include a number of nucleotides in the reaction mixture such that the number of PCR amplification products produced is more related to the amount of nucleotides than to the amount of DNA in the original sample. It may include limiting the amount. Microwells with a large amount of target DNA run out of nucleotides early in the cycling step, whereas microwells with a small amount of target DNA run out of nucleotides later in the cycling step, but with approximately the same amount An amplification product is produced.

  Multiple embodiments to limit variation in the amount of PCR product across the well are available for microorganisms growing in the well so that the cells self-replicate until the medium is exhausted and stops replicating Limiting the amount of nourishment nutrients.

  Multiple embodiments for limiting variation in the amount of PCR product across the well place a dye in each well that identifies the PCR product so that the signal shines more as more PCR product is produced Can include. During each PCR cycle, dye brightness is measured and managed, and once the desired signal intensity is observed, the sample can be removed from the well.

  Multiple embodiments for limiting variation in the amount of PCR product across wells can be achieved with oligos complementary to barcodes specific to each well to selectively hybridize amplified DNA from each well. It may include using a mixture of covered hybrid-producing beads. Once the beads are saturated, unbound DNA is washed away and bound DNA is released from the beads. The amount of DNA from each well is then normalized with the bead saturation limit.

  Embodiments for limiting variation in the amount of PCR product across a well include that fast-growing microorganisms rapidly fill the well and stop replicating, slower-growing microorganisms gradually fill the well, and It may include warming the chip for culture for an extended period of time to stop growth once the same number of cells are in the well.

  In embodiments, the use of barcoded primers and next generation sequencing (NGS) within the context of the chip format and method allows which species are grown in which wells on the high density microchip. Can be used to identify When approximately the same number of bacteria occupy each microwell in the chip, the signal from each well of the NGS data can be almost the same.

  For example, in a typical NGS run that yields 12 million sequence reads, 24 chips each having 10,000 microwells are sequenced in the run, and if there are the same number of bacteria per well, 1 well On average there will be 50 readings.

  However, when different bacteria grow at different rates, multiple wells will have a small number of bacteria and multiple wells will have a large number of bacteria. This is distorted when NGS runs so that wells with a small number of bacteria are not detected by NGS analysis.

This is why in a typical NGS implementation that yields 12 million sequence reads, each chip has 10,000 microwells, half of those microwells containing 100 times more bacteria than the other half. Suppose the 24 chips in it are sequenced per run. The probability of detecting bacteria in wells that grow slowly is significantly reduced. in this case,
(10,000x24) / 2x100 = 12,000,000 (1)
(10,000x24) / 2 = 120,000 (2)

Low frequency wells represent 1% of the total. Thus, of the 12,000,000 readings in one NGS run, 120,000 are from low frequency wells, ie, an average of 1 reading per well.
In order to minimize the impact of this phenomenon, new methods need to be developed to help homogenize the amount of PCR product across the wells so that all wells are detected in the NGS run.

(Microwell chip based on silicon for isolation, growth, selection and analysis of microorganisms)
The microfabricated device or chip can be at least partially composed of silicon instead of or in addition to plastic, glass and / or polymer, taking into account electrical measurements per well. For example, the walls of each well are insulated to create a microcapacitor. In another example, the FET in each well can be isolated such that the gate surface is exposed to the well contents. Instead of a tip that uses pure silicon only, a thin metal layer may be produced on the top surface of an existing tip by plating, vapor deposition, and / or arc / flame spraying. This can be expected to add more functionality, use cheaper and / or cleaner alternative manufacturing methods, and / or reduce the size and weight of handheld and / or portable devices.

  Embodiments may consider the case of measuring growth by electrical measurement. Periodic measurement of impedance can be applied to the measurement of microbial (eg, bacterial) growth. For example, the impedance at both ends of a tube containing E. coli is the cell number of that tube in Ur et al., “Impedance Monitoring of Bacterial Activity”, 8: 1 J. Med. Microbiology 19-28 (1975). It is compared. This article is incorporated herein by reference in its entirety. In order to prove that this effect is common, measurements may be made on other types of bacteria including Judemonas, Klebsiella and Streptococcus. To test growth conditions across different formulations, the wells may be filled with different media.

  Embodiments can allow for sorting by electrical measurements. Electrical measurements can be made for each well that allows for sorting. One example was pH. There are a number of different ways to obtain a pH dependent response from the gates of in-well devices including ISFETs, pH meters, and the like. Well arrays with embedded pH sensors can electrically determine which wells contain microorganisms that produce acidic or basic metabolites. A simple example is a test targeting lactic acid production from lactose. The bacteria are diluted and distributed into the wells, grown and then fed with lactose. Wells that record a drop in pH contain microorganisms that can break down lactose into lactic acid.

Embodiments can envision electrical measurements of redox probes. Another way to influence electrical measurements is to see how bacteria in the well affect known redox probes. Basically, systems with well-defined responses can be measured in the presence of bacteria, and deviations from expected behavior can be attributed to bacterial samples. A typical redox probe is something similar to ferricyanide, [Fe (CN) 6] ^{3- / 4-} . The reduction of ferricyanide to ferrocyanide is very well characterized, especially as a small behavioral change around the electron transfer from the electrode can be found. This system is “unlabeled” because it can detect bacteria themselves without having to modify them directly.

  An antibody capable of recognizing a microorganism (for example, E. coli) can be immobilized on the ITO electrode. Electron transfer resistance from the electrode to the ferricyanide-containing solution can be measured. E. coli bound to the electrode surface increases the resistance in proportion to the E. coli concentration on the surface. This is an example of a group of measurements that can detect a specific type of organism or metabolite using a redox probe.

  The work provided by silicon (or at least metal or metal-coated plastic) is the ability to produce cheaper chips (eg, by piggybacking on existing technology), comprehensive detection capabilities that enable smaller and / or portable versions, and other materials (eg, , LCR, CV, etc.) provide various advantages, including integration of detection modes with newly discovered chips into existing devices, a combination of electrical measurements and sequencing. These advantages will benefit any customer using an interferometric detection method.

(Peelable barrier protecting well-specific chemicals on the chip)
21A-21E are lists illustrating chips having well-specific chemical agents according to embodiments. FIG. 21A shows a device or chip made by microfabrication and having a plurality of microwells. In FIG. 21B, microwell-specific chemical agents are assigned to each microwell on the chip. In FIG. 21C, the sealant covers the microwell-specific chemicals within each microwell of the chip, thereby preventing their interaction with further additives to the wells. Yes. In FIG. 21D, the sample is filled and the experiment is performed on the sample in the macrowell. In FIG. 21E, a trigger (eg, heat) releases the microwell-specific chemical agent for interaction with the sample in the well.

  Microwell chips are manufactured, cleaned and / or their surfaces are treated. Specific chemical agents are prepared separately and then, for example, using methods and / or instruments that allow a set of specific chemicals to be directed to a specific well (or multiple wells), Put in the well. Immediately thereafter, sealants may be applied to protect the various chemical agents from removal and release and / or interference from the environment and / or by some limited external trigger.

Microfabricated devices or chips can be manufactured with a specific design, for example, cleaned and / or surface treated to enhance wetting. PCR primers can be printed into specific wells or dotted with pins. The chips can be dried, after which the wax layer can be deposited by evaporation of the ethanol solution. The optimal concentration is about 1% v / v. The molten wax may be applied directly, or an aqueous wax solution or an alcohol solution may be sprayed. Alternatively, spin coating or vapor deposition may be used. Various waxes can be used, examples of which include glyceryl stearate, cetearyl alcohol, 1-hexadecanol, glyceryl stearate, ceteares-20 (CAS Registry No. 68439-49) with or without polyethylene glycol. -6), and multiple commercial products such as Lotionpro ^™ 165 (available from Lotioncrafter®, Eastsound, Washington) and Polawax ^™ (available from Croda, Inc., Edison, NJ) It is done. The underlying chemical agent can be released later, for example by heating until the wax melts. For these compositions, the heating is between 50 ° C and 70 ° C. It is important that the heating temperature be low enough not to damage any chemical components or to boil our aqueous solution.

  An important concept is a well-specific chemical agent that is separated from the chip until the experimenter releases the trigger. This method can be used for bar-coding within a well, but can also be applied across a wider range of different problems. Various chemical agents useful for sealing on the chip include, but are not limited to, antibiotics, fluorescent materials, dyes, PCR primers, dissolution promoters, antibodies, reagents for various metabolites, etc. is not. Wax is a good means to seal things that can later be released by heating, but other materials are used to seal and release against exposure, sonication and / or some other trigger. Also good. This is superior to simply adding a reagent to the chip because it controls each well. A similar effect may be achieved by printing chemical agents into the wells after performing a microbial test, but this is a matter of time (printing steps can take a day) and If each well is clogged individually after the microorganisms are present on the chip, this introduces the fact that it is impossible to have all wells acted on in the same amount of time. With the release mechanism, all wells can be affected simultaneously. In one example, the wax may be deposited on the chip by solvent casting.

(Isolated microwells for easy and / or relative abundance control)
A high density chip device may comprise a surface having high density microwells. Microorganisms, or other cell types, from the microbiota sample can be diluted and applied to the device such that the well contains approximately one microorganism or cell per occupied well. These microwells can be sealed with a semi-permeable membrane that allows diffusion of nutrients into the microwells but prevents all or at least some of the microorganisms or cells from moving out of the microwells.

  Microbial or cellular samples can be prepared and then encapsulated in a chip using a membrane that is impermeable or gas permeable only. Since no liquid reservoir is placed on top of the chip or on the membrane, only the nutrients in the wells at the time of sealing can be used to help the growth of microorganisms or cells in the microwells. Two grounds for this feature are: (1) the simplicity of the structure and device workflow does not require a semi-permeable membrane or reservoir, no nutrients have to be added, and There are less fears of contamination, and (2) reducing the relative abundance of premature organisms by limiting the availability of nutrients. For samples containing multiple premature organisms and multiple late organisms, premature organisms quickly become a source limit in their microwells and stop growing or slow, while the adult organisms continue to grow. This ensures that the adults are shown with a higher relative abundance in the population of microorganisms across the chip than in the case where there is no limit on the nutrient content of the adults. This is important for downstream processing during all sequencing on the chip. It also provides a better detection limit for rare species because the propagation of rare species is not accelerated by premature species to the point of limiting the performance of their detection system.

  Current methods attempt to propagate all species regardless of whether they are native or late. This has the inevitable consequence that the early adult species swept the community and only increased relative abundance over time. Many types of downstream analysis, such as sequencing or fluorescence sorting, cannot analyze all species except those above a certain limited relative abundance within a given solid county. If the goal is to detect rare species while retaining diversity, then it is necessary to limit premature species in some way.

As an example to illustrate this idea, a simple example of a sample containing two species, ie one species doubles every day and the other species doubles every week, as shown in Table 6. Let's consider. Assuming that the late adult species is initially rare, with a relative abundance of 5%, it becomes very rare as both species grow.

If premature species compete for nutrients and / or are limited by the physical space for propagation, then the relative abundance of late adult species begins to increase after some time, as shown in Table 7. Yes.

(High-density microfabricated array for biobank cells)
Biobanks are designed to pave the way for researchers to obtain large numbers of samples from large populations to drive certain types of research, such as the discovery of disease-related biomarkers. The state of the art in biobanking provides for samples to be stored in tubes or low density plate forms, such as 96-well or 384-well plates. This works when the number of samples to be charged is relatively small and the samples themselves are discrete, isolated solid groups. Existing approaches to biobanking can be complex and inefficient when storing samples such as microbial samples, where the number of samples can be large and the number of different species or variants within each sample is one. It can range from hundreds to thousands or even millions per sample. Using existing methods, a laborious isolation protocol must be implemented to separate individual species or variants either prior to or following the storage step to obtain the desired species or variant. Don't be.

  The systems, kits, devices, and methodologies described herein may be applied to biobank cells, microorganisms, viruses, and other biological entities. A high density chip device comprising a surface with high density microwells can have thousands, hundreds of thousands or millions of microwells per chip. For example, microorganisms (or other biological entities such as different types of cells or viruses) from the microbiota sample are diluted and applied to the device so that the wells contain approximately one microorganism per occupied well. Is done. The chip is then warmed so that the microorganism is replicated in the well and the resulting population is a single species. A nucleic acid location indexing system can be used to determine what species is present in each well. This indexing system may require that each well has pre-loaded PCR primers. These PCR primers may contain a primer sequence directed to a specific genetic element (eg, 16S) of a microbial gene that provides an addressing barcode that identifies the well and species information or targets the desired gene sequence . After incubation, microbial DNA is released and PCR primers amplify the target bacterial DNA region. Amplification products from various wells on the chip can be pooled and subsequently decoded by next generation sequencing.

  The use of high-density microfabricated chips for biobanking allows multiple species within each sample to be stored as separate populations without the need to perform laborious isolation protocols either before or after storage. Or prepare for a variant. The use of DNA location indexing systems or application specific testing methods allows a simpler general approach to identifying the genetic markers or characteristics of the contents of each microwell, such as species information, Give information. In addition, chip devices provide for a very space efficient way of storing cell segregation populations. For example, a single microscope slide size tip with 100,000 wells occupies much less space than a corresponding conventional storage configuration. The chip format also handles the database of subject (e.g. patient) information by properly recording and managing samples and / or by accommodating many different samples from a single object on a single chip. Can be useful for.

  In order to line up cells using this device, the cells can be placed and / or positioned in microwells of the device. Cells in the microwells can be treated to improve the storage effect and then placed in appropriate storage conditions. For example, cells can be treated with agents such as glycerol to improve the effects of freezing. The chips can then be placed under appropriate storage conditions such as a freezer. The cells may be dehydrated, lyophilized and / or vacuum lyophilized, and the chip may be placed under appropriate conditions for dry cell protection. Additional structures may be added to the chip to further enhance its usefulness as a storage device. For example, a membrane or other structural component may be placed on the top surface of the chip to seal at least some of the wells prior to storage.

  The chip can be loaded with cells such that each portion of the microwell on the chip is occupied by approximately one cell. The chip is warmed in anticipation of cell replication. The chip and its contents are substantially replicated, and either the original chip or the replicated chip uses the cell or species or genetic marker present in each well of the chip (eg, using the position indexing system described above). Used to identify). The chip can be processed and / or stored at appropriate conditions. The duplication step and / or identification step may be performed after storage rather than before storage.

  One or more cells from each of a set of pre-existing isolated strains can be placed in separate microwells on the chip. The location of the microwell where each strain or cell type is located is recorded, and the chip can be processed and / or stored under appropriate conditions.

One chip can be created in which the storage chemistry is sequestered directly under the wax barrier in each well. The isolate can be sealed inside the chip and allowed to grow and then protected from later days by heat-induced release of preservatives prior to banking.
Such devices can be used to store / biobank mixed microbial community samples such as microbial community samples from soil, human intestines, seawater, oral cavity, skin, and the like. The chip can be used to store other types of biological entities such as fungi, archaea, human cells (including germ cells), animal cells and viruses.

  The DNA position labeling system can be used to create information about the contents of each well across all types of biological entities. The entire chip can be tested for the desired activity using specialized analytical methods such as antibody or substrate analysis. For example, a chip with a bank population of T cells can be tested for acquired immune activity.

  In one illustrative example, human fecal microbiota samples may be collected from study participants. Individual faecal microorganisms can be biobanked on the chip to maintain a record of the individual's microflora as well as as a sample of the microflora for later use as a source of target microorganisms. In another example, a sample of a mixed population of T cells or other acquired immune cells is taken from an individual during clinical or research studies or as part of a treatment workflow (eg, treatment using cells treated in vitro). May be examined. In yet another example, soil biomes may be stored in cooperation with seed banking.

(High resolution sampling)
Highly accurate / precise sampling devices or systems can be designed to perform various sampling functions from and / or to the microwells on the microfabricated chip described above. The target substrate or tip can be a microscope slide type (about 25 mm × 75 mm × 1 mm) with a mechanism injection molded on one surface. The microwells can be arranged in a grid pattern with a well-less edge of about 4-8 mm around the sides of the chip. Well size and spacing can be determined based on collector performance. The microwells can be square with a dimension along each side of about 25 μm to about 200 μm, with a spacing of about 25 μm to about 100 μm between the well ends. The microwells may alternatively be circular or hexagonal. The depth of the well can be from about 25 μm to about 100 μm. For example, a 75 mm × 25 mm slide with 7 mm edge, 100 μm square wells with 100 micron spacing between well ends would have about 16,775 microwells.

  Highly accurate / precise sampling system to perform various sampling functions from and / or for part of the membrane corresponding to the microwell on the microfabricated chip as described above Can be designed to. The membrane may be a thin sheet that has already been used to encapsulate the growing bacteria into the microwells. When peeled from the chip, the membrane can retain indentations of the microwell array as well as bacterial samples on its surface after separation from the chip. Thus, the release membrane can function as a replica of bacteria grown on the chip.

  The high resolution sampler can receive data input from the user. The input includes at least one pair of chip microwell coordinates, so that the collector can sample from and / or to at least one input pair coordinate. Mechanical sterilization operations can be performed between cycles. The high resolution sampler may be operable in an anaerobic chamber.

  The high resolution collector system receives the chip and can be adapted to the chip using, for example, a reference label and / or a reference well. The collector can collect, for example, cells grown in microwells on the chip into 96-well or 384-well plates containing, for example, culture medium. The collector may include a single collection pin or multiple collection pins.

  The high resolution collector system receives the membrane and can use the reference well label on the membrane, for example, to align the collector to the membrane. The harvester can, for example, harvest cells from the membrane into 96-well or 384-well plates containing, for example, culture medium. The collection pin can have a variety of shapes (eg, mushroom shape) and / or a collection surface (eg, texture) from the membrane. The system may also include one or more mechanisms (eg, floating pins and / or vacuum) for holding and / or flattening the membrane.

  A high resolution sampler system may allow chip replication via chip-to-chip movement. The collector may receive and align the first chip based on the reference label and / or the reference well. The collector may also receive and align a second tip based on the reference label and / or the reference well. The harvester can, for example, transfer cells grown in the microwells on the first chip to the microwells on the second chip.

  A high-throughput system for automatically harvesting a target species among multiple species of at least one biological entity cultured on a microfabricated device may include a port that accepts the microfabricated device. Microfabricated devices define a high density array of microwells. Each microwell of the high density array of microwells is configured to isolate and culture at least one species of at least one biological entity and includes at least one of a plurality of specific tags. Each tag of the plurality of specific tags includes a nucleic acid molecule that correlates with a target-specific nucleotide sequence for annealing to the at least one biological entity as well as at least one microwell of a high density array of microwells. It shall include the relevant position-specific nucleotide sequence. The system also incorporates a high resolution collection device with at least one projection for collecting at least one biological entity from at least one microwell of a high density array of microwells. The system further incorporates an input device for receiving an indication of at least one target-specific nucleotide sequence and at least one processing unit communicatively connected to the input device and the high resolution collection device. The at least one processing device obtains at least one target-specific nucleotide sequence from the input device, compares the at least one target-specific nucleotide sequence to a plurality of specific tags, and based on the comparison, the high density macrowell High resolution to determine at least one microwell containing a target species in the array and to collect at least one biological entity from at least one determined microwell in the high density array of macrowells Control the collection device.

  A high-throughput system is disclosed for automatically harvesting a target species among a plurality of species of at least one biological entity cultured on a microfabricated device. The microfabricated device defines a high density array of microwells, and each microwell of the microwell high density array is associated with at least one unique primer of the plurality of unique primers. The system includes a port that receives a membrane that has been removed from a microfabricated device, the membrane of the microwell high density array to retain at least one biological entity within the microwell high density array. Since each microwell was sealed, a portion of at least one biological entity corresponding to the high density array of microwells remains attached to the membrane after removal of the membrane. The system also includes a high resolution collection device including at least one protrusion for collecting at least one biological entity from at least one membrane location corresponding to at least one microwell of a high density array of microwells; It incorporates an input device that receives an indication of at least one target-specific nucleotide sequence that is associated with the target species, and at least one processing unit that is communicatively connected to the input device and the high-resolution collection device. At least one processing unit obtains at least one target-specific nucleotide sequence representation from the input device, compares the at least one target-specific nucleotide sequence to a plurality of specific tags, and has a target species based on the comparison Determining at least one membrane location corresponding to at least one microwell of the high density array of microwells and taking a portion of the at least one biological entity from the at least one determined membrane location; Control the resolution sampling device.

  The above embodiments can be implemented in any of a great many ways. For example, the embodiments may be realized using hardware, software, or a combination thereof. When implemented in software, the software code may be executed on any suitable processor or collection of processors, whether provided on a single computer or distributed to multiple computers.

  Moreover, the computer may be embodied in a device that is not generally considered a computer, including a personal digital assistant (PDA), a smartphone, or any other suitable portable or fixed electronic device, but that has appropriate processing capabilities.

  Furthermore, the computer may have one or more input and output devices. These devices can be used, among other things, to provide a user interface. Examples of output devices that can be used to provide a user interface include a printer or display screen for visual presentation of output and a speaker or other audio generating device for audible presentation of output. Examples of input devices that can be used for user interfaces include keyboards and positioning devices such as mice, touchpads and digitizing tablets. As another example, a computer may receive input information via speech recognition or other listening methods.

  Such computers are interconnected by one or more information communication networks of any suitable form including regional or wide area information communication networks, such as, for example, corporate communication networks and intelligent networks (IN) or the Internet. obtain. Such an information communication network can be based on any suitable science and technology, can operate according to any suitable protocol, and can include a wireless communication network, a wired communication network, or an optical fiber communication network.

  The various methods or steps outlined herein can be encoded as software executable on one or more processing devices using any one of a variety of operating systems or platforms. In addition, such software can be written using a number of suitable programming languages and / or programming or scripting means, and executable machine language code or code executed on a framework or virtual machine. It can also be converted as an intermediate code.

(Determination of bacterial group relationship)
In embodiments, the systems, surely apparatus and methods described above can be used to determine bacterial group relationships. Microwell arrays on a microfabricated device (or chip) having the position index system described herein (eg, each microwell oligonucleotide containing code information that identifies those particular microwells, as well as nucleic acid sequences) Can be used to analyze sympatric or other types of mutants or species relationships in microbiome samples in a massively parallel manner.

  In accordance with embodiments of the present invention, a method for analyzing a sample containing a population of biological entities is provided. The method utilizes at least one microfabricated device having a top surface defining a microwell array, each of the plurality of microwells of the array of microwells being (1) one or more objects that may be present in the microwell A tag-specific nucleic acid sequence with a target-specific nucleotide sequence for annealing to a target nucleic acid fragment of a biological entity, and (2) a position-specific nucleotide sequence for locating a microwell on a microfabricated device Include as. A certain amount of nutrient is also filled in each of the plurality of microwells. This may be done as a separate step, or the nutrient and biological entity may be put together at the same time. A microfabricated device having a filled microwell is incubated under predetermined conditions. Then, for example, by performing PCR amplification on selected genetic material (eg, bacterial, fungal, or eukaryotic genomic DNA) of cells of the biological entity, each individual of the plurality of microwells. Amplification is performed on-chip in the microwell. When amplification is performed, amplification may not be successful in each microwell. The first amplicon is collected / pooled from a plurality of microwells and sequenced to obtain sequencing data. Based on the sequencing data and the unique tag nucleic acid molecule contained in each of the plurality of microwells, biological entities present in at least a subset of the plurality of microwells are identified. In some embodiments, between at least two different types of biological entities in the population of biological entities included in the sample based on the identification of biological entities present in each of the plurality of microwells. The presence or absence of the relationship is determined. A relationship is either a subordinate relationship, a symbiotic relationship, or a destructive relationship. The presence or absence of a relationship can be determined by comparing across multiple microwells what type of biological entity is present or absent in the same microwell after incubation.

  For example, cells isolated from a complex microbiome sample (ie, containing one or more species or variants) are microfabricated with appropriate dilution and distribution methods that yield an average of two or more cells per microwell Can be dispensed into microwells of the device. The dispensing method may be, for example, by applying a solution containing the microorganisms on the array so that the microorganisms are randomly distributed in the microwells. The concentration of microorganisms in solution can be adjusted to target the desired average number or distribution of bacteria per microwell. Alternatively, the microorganism may be directly placed in the microwell using an apparatus such as a cell sorter.

  The target average number of cells per microwell can vary depending on the number of microorganisms in the sample and the dependency complexity being assessed. For example, it is 2 cells per microwell, 5 cells per microwell, 10 cells per microwell, 50 cells per microwell, 100 cells per microwell, or other number of cells per microwell obtain. In some embodiments, the average number of cells per microwell is between 2 and 20. In other embodiments, the average number of cells per microwell can be between 5 and 50, between 10 and 100, and the like. There are microwells that contain no cells, microwells that contain one cell, and microwells that contain more than the required number of cells.

  In an embodiment of the method, the population of biological entities in the sample comprises bacteria. Bacteria can include bacteria of different strains, different species, or different genera. A population of biological entities can include a collection of microorganisms that are naturally present in a particular environment. For example, a biological entity can be a collection of microorganisms obtained from human stool, human intestine, human skin, human nasal cavity, vagina, soil, rhizosphere, water, and the like. In some other embodiments, the biological entity may comprise a eukaryotic cell.

  After being placed on the microfabricated device, the cells can be cultured by incubating the microfabricated device under appropriate conditions. Incubation can be carried out in various ways. For example, a membrane can be applied to the top surface of the microfabricated device to hold the cells loaded in the microwell prior to incubation. Nutrients can be pre-filled into microwells and retained by the membrane. In some embodiments, nutrients can be filled into reservoirs provided on the membrane and outside the microwell. The membrane is permeable to nutrients and allows nutrients to move from the reservoir to the microwells.

  After incubation, the microfabricated device may be replicated or divided as described above to make a replica of the contents of the microwell by removing a small amount of sample from each microwell. Then with appropriate indexing so that on-chip PCR or other nucleic acid amplification means can be performed to generate a list of cell contents (genus, species, strain, variant, etc.) for each microwell for analysis Amplification products or amplicon libraries can be generated.

  Potential dependencies that exist within a microbiome by observing which genus, species, or other types of microorganisms grow together in the same microwell across an array of microfabricated microwells Or a relationship can be determined. For example, the relationship can be a subordinate relationship, a symbiotic relationship, a restraint relationship, or an independence. For illustration purposes, a dependency between two species is that one or both species depend on the other to survive, thrive, or multiply. If both species are dependent on each other to survive, thrive, or multiply, they are considered to be interdependent. If the first species is dependent on the second species, it will most likely only detect growth in the presence of the second species. If the first and second species depend on each other, neither species will be found growing in the absence of the other species. A symbiotic relationship between two species means that the first species can survive, thrive or proliferate in the absence of the second species, but grow or proliferate more advantageously in the presence of the second species. That is, the repressive or destructive relationship between two species is that one species inhibits or kills the growth of the second species and the first species is inhibited by the second species If the first species is not detected, or is detected only at a low level whenever the second species is present, the independence (or lack of relationship, lack of relationship) between the two species It means a situation where both the first and second species can grow well together and separately. It will be detected that both the first and second species are growing together and separately.

  Various abundance thresholds can be used to analyze the results from the microwells to determine the potential relationship between the two species. For example, a first species of less than 1% may be considered equal to zero or a first species of less than 5% means inhibition. The exact nature and level of the cut-off will vary between different biological systems and compositions. This method can be used to analyze interspecies relationships across microbiomes containing hundreds or thousands of different components / species / variants.

  Consider a group with two elements A and B. They are filled into chips, grown, indexed and arranged. Sequence data can be studied to determine which species grow with which other species, thereby providing insight into their relationships and dependencies. If A and B can coexist, the expected results are microwells containing A without B, those containing B without A, and microwells containing both A and B. When A destroys B, there are multiple instances of A, multiple instances of B, and minimal or no instances of A + B. A and B are in a true symbiotic relationship, there are multiple examples of A + B, and there may be minimal or no examples of A or B.

  More specifically, to determine the relationship between two biological entities of interest in a microbiota sample, for example, the cells of microorganism A and another microorganism (microorganism B, microorganism C, etc.), the following example procedure Can be used. This procedure is exemplary only and does not limit the invention.

1. Take a sample of soil.
2. Microbial cells in the soil are separated from the rest of the soil to produce a suspension of microbial cells. This can be done by filtration and centrifugation using a medium such as Nycodenz.
3. Count the microbial cells in suspension using techniques such as counting cells in a cell counting slide (such as a hemocytometer) under a microscope or using a flow cytometer.
4). Take the microfabricated device described herein and incorporate in each microwell i) a barcode nucleotide sequence (eg, microwell row and column number, or other relative position) specific to the individual microwell Preload with PCR primers. (Ii) Nucleotide sequences targeting the bacterial 16S rRNA gene region. (For analysis of microbiomes, including microorganisms, nucleotide sequences can target the ITS gene region) Primers can be protected by a wax coating. Both primer and wax can be printed on the microwells using a high resolution printing device.
5). The cell suspension is diluted so that the expected (or average) number of cells per occupied microwell is 5 when filled into the microfabricated device of the present invention. The cells can be suspended in a suitable growth medium.
6). Fill the microwells by pipetting the diluted cell suspension onto the surface of the microfabricated device so that all the microwells are covered with liquid. Part of the bacterial suspension enters the microwell.
7). (A) Place an impermeable or gas permeable membrane over the microwell, or (b) place a permeable membrane over the microwell, and then place a nutrient reservoir containing nutrients.
8). Incubate the microfabricated device for a period suitable for the species to be tested and for appropriate conditions (temperature, humidity, atmosphere, etc.).
9. The microfabricated device is replicated using the methods described herein. Add membrane to one or both replicates and incubate several more as needed.
10. Take a duplicate of the chip containing the PCR primer, soak in liquid nitrogen, then remove from the liquid nitrogen, then remove the membrane, add the PCR mixture to the microwell, cover the microwell with oil, and bacteria in the flatbed PCR thermocycler On-chip PCR is performed by amplifying the genomic DNA.
11. Pool PCR products or amplicons from microwells on the chip and add sequencing adapters and sample-specific barcodes for the second PCR reaction.
12 Sequence amplicons in next-generation sequencing systems such as Illumina Miseq. This can give the reading of most position-specific barcodes on the microwell, and the 16S sequence will indicate which microorganism is growing in which microwell. Given the nature of the 16S analysis, this information will generally be at the genus or species level.
13. The results are analyzed to determine which species or genus has grown in each microwell.
14 Identify wells containing microbe A. For each microwell containing microorganism A, identify those in which other species are growing in the same microwell. Since each well has a limited number of species, it is possible to identify which additional species can support the growth of microorganism A. For example, the result is that microorganism A grows only in the presence of microorganism B, microorganism C or microorganism D.
15. The experiment can be repeated using higher dilution levels to target as few as two microorganisms per well. This can provide information for confirmation.
The above methods are: i) which species always grow with other species (including dependencies (including interdependencies)), ii) species that never grow with certain other species (destructive or Indicating a suppression relationship). ), Iii) A map of the entire microbiome may be created for which species can grow independently of other species. This data may be used to create a relationship map across the microbiome to identify clusters of microorganisms that share interdependencies. This information can be independent of, for example, which microorganisms in a microbiome sample can be easily cultured (and because they grow independently) and which microorganisms depend on other species for important growth factors. And may be used to predict whether it may be difficult to culture.

  The above method may also be used as a discovery tool for new antibiotics. For example, microfabricated chips can be produced in microwells containing only one species, for example by diluting to a predetermined concentration or by using a cell sorter to apply microorganisms in solution to the microwells on the array. Both are filled so that they are not mathematically possible. If any microwell contains only one species, that species is likely to produce antibiotics (ie, other species that were previously in the microwells are due to antibiotics secreted by the living species) It seems to have been killed). A viable sample of putative antibiotic producers can be amplified and further tested through chip splitting.

  Different microbial growth media are used on the array to underlie the effects of media formulations (or nutrients) on the relationship between bacterial variants or species, eg, promoting or delaying interdependencies, and such effects Can understand chemistry. For example, multiple microfabricated devices containing different media formulations or nutrients can be loaded with microorganisms from a microflora sample and processed as outlined above. Differences in growth-dependent maps observed between arrays will show how each medium affects sympathetic relationships or other interactions between different bacterial variants or species. Medium X can be formulated to be free of free amino acids. Medium Y is the same as medium X but can be formulated to contain free amino acids. It is assumed that species A grows only in the same microwells as species B when medium X is used, while species A can be grown independently of species B when medium Y is used. Using multiple formulations of media and analyzing across all microwells, a map of symbiotic relationships and indicators of the underlying nature of these relationships can be developed for the microbiome.

  Alternatively, different media formulations may be added to microwells placed in different subareas of a single microfabricated device and inoculated with the particular microbial species or microbiome under study. The observed growth dependence differences from different media provide insight into the underlying nature of the relationship.

  In addition, the effect of different media formulations on bacterial relationships as well as individual bacterial growth may be determined. For example, relationships within the desired microflora may be studied according to the methods described herein, and microorganism G is determined to grow only in the presence of microorganism K using a given growth medium. The microbiome may be reanalyzed using several different media on different microfabricated chips. For a particular medium formulation, it is determined that microorganism G can grow in the absence of microorganism K. This particular media formulation is then broken down and various components are used in further experiments to determine which components promote the growth of microorganism G in the absence of microorganism K.

  Alternatively, the above can be accomplished as follows: relationships within the microbiome of interest are studied, and microorganism G is determined to grow only in the presence of microorganism K using a given medium. . The array is then made so that different media formulations are placed in different microwells on the array. This can be done, for example, by feeding different media formulations to different wells using a very accurate printing device such as an ink jet printer. The array is then loaded with a mixture of microorganisms G and K.

  Then, using the above position index system, determine whether i) only G grows, ii) only K grows, iii) neither G nor K grow, or iv) both G and K grow can do. Such growth information correlates with the media recipe and is used to infer which components are important in the growth relationship.

  Theoretically, the above studies on microbial interactions can be performed on traditional platforms (eg, Petri dishes), but by using a microfabricated chip with an array of microwells of the present invention, Although studies can be conducted in massively parallel and highly multiplexed formats, it does not require a priori knowledge of how each species behaves. A single experiment or a small number of experiments may allow identification of multiple relationship dependencies in complex microbiomes. If a relationship of interest is found, coupled with the ability to sample each well (by duplicating the contents of the microwells) before obtaining sequence data to build the relationship map, such a relationship is discovered. The sample has already been duplicated. It makes it possible to reserve a viable sample of microorganisms for further experiments.

In the examples below, each microwell on a panel of 2500 (50 x 50) microwells of microfabricated chips (each square with 100 μm x 100 μm and 100 μm deep edges) functions as a PCR amplification primer Fill the 16S bacterial gene region with your own tag oligo. Each tag oligo includes a barcode sequence to identify a specific location in the microwell filled with the tag oligo. Since the microwells are arranged in a grid, the bar code array herein includes a subcolumn for identifying the microwell columns and another subcolumn for identifying the rows.
The method used is shown below.

Chip PCR protocol Dilute bacteria with TSB medium 1:10.
2. Place 3 μL of bacteria in each panel of the chip and seal with a PCR sealer.
3. Incubate the chip with bacteria overnight in a 30 ° C incubator.
4). Immerse the tip in liquid nitrogen to facilitate dissolution.
5). Remove the seal.
6. Fill with 3 μL of chip PCR buffer and seal the chip with PCR sealer.
7). Fill the Life Tech QuantStudio 3D digital PCR system with chip and perform chip PCR.
PCR amplification protocol: 96 ° C for 12 minutes;
39 cycles of 60 ° C for 2 minutes and 98 ° C for 40 seconds:
60 ° C for 2 minutes;
Maintain 10 ° C.
8). NGS library construction: Prepare a NGS library by performing a second PCR amplification to add a sample index and NGS adapter for each chip sample. After confirming the presence of the correct product by gel electrophoresis, the amplicon is purified with Kapa purification beads and a final aliquot of 25 μL amplicon product is collected for NGS.

Illumina MiSeq and bioinformatics MiSeq was loaded with 10 pM DNA library and NGS was performed.
2. Decode data using unique tag information to identify the location of each NGS read for each microwell of the microfabricated device. NGS reading data is thresholded to 5000 readings per well.

  Fill a microwell panel with 8 mock groups of live bacteria and incubate overnight. The mock group consists of Acinetobacter calcoaceticus, Bacillus cereus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcens and Staphylococcus warneri. After performing chip PCR and NGS as described above, the NGS data indicates that there were approximately 7,514,030 total NGS readings. Bacteria were detected in 1503 microwells (about 60% of all microwells in the panel). In 92% of these microwells, multiple bacterial genera were detected. A single bacterial genus was detected in 108 microwells, most of which were E. coli. Some microwells contained multiple species, each of which was E. coli. It was the most common species among these microwells. One explanation is that E. coli grows rapidly and occupies most of the well. To understand the distribution of the number of bacterial genera grown in each microwell, the data are summarized in FIG. 108 wells with single genus bacteria detected, 426 wells with 2 bacterial genus detected, 334 wells with 3 genus detected, 635 wells with 4 or more genus detected did.

  While various embodiments of the invention have been described and demonstrated herein, those skilled in the art will recognize that various other implementations may be performed to perform the functions and obtain the above results and / or one or more advantages. Means and structures should readily be envisioned, and each such modification and / or modification is considered to be within the scope of the embodiments of the invention described herein. More generally, one skilled in the art means that all parameters, dimensions, materials and configurations described herein are representative, and actual parameters, dimensions and / or configurations are It should be readily apparent that the teachings of the present invention depend on the particular application or applications in which they are utilized. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Therefore, the foregoing embodiments have been presented by way of example only and, within the scope of the appended claims and their equivalents, inventions in different ways than those specifically described and claimed. It should be understood that the embodiments may be implemented. Embodiments of the present invention are directed to the individual functions, systems, articles, materials, kits and / or methods described herein. Further, if such functions, systems, articles, materials, kits and / or methods are not in conflict with each other, combinations of two or more of those functions, systems, articles, materials, kits and / or methods Are included within the scope of the present invention.

  Moreover, the various inventive concepts are embodied as one or more methods, examples of which are provided. The operations performed as one ring of the method can be ordered in any suitable manner. Thus, embodiments may be configured in which operations are performed in a different order than illustrated, including multiple operations performed at the same time, even if shown as sequential operations in the illustrated embodiments. It's okay.

  The full text of each of all works, patent applications, patents, and other references mentioned herein is hereby incorporated by reference.

  All definitions should be understood to govern the dictionary definitions, definitions in the literature incorporated as references and / or the ordinary meaning of the defined terms, as clarified and used in this document. It is.

  The indefinite articles "a" and "an" used in the specification and claims of this application are understood to mean "at least one" if they are "one" or if no number is explicitly specified. It should be.

  As used in the specification and claims of this application, the phrase “and / or” includes the elements so conjoined, ie, elements that are conjugate in some cases and separated in others. Should be understood to mean “one or both”. Numerous elements listed with “and / or” should be construed as well, ie, one or more of the elements are so associated. In addition to the elements specifically identified by the phrase “and / or”, other elements may optionally be present whether or not related to the identified elements. Thus, as a non-limiting example, a reference to “A and / or B” may be used in conjunction with an unrestricted phrase such as “includes” in certain embodiments (elements other than B are optional). Only A can be referenced, in other embodiments only B can be referenced (although elements other than A are optionally included), and in other embodiments A and B (although other elements are optionally included) Both of them can be referred to.

  "Or" as used in the specification and claims of this application should be understood to have the same meaning as "and / or" as defined above. For example, when separating an item in a list, “or” or “and / or” also includes everything, that is, not only include at least one of the many or list elements, but also include one or more. It shall be construed to include items that are included and optionally not included in the additional list. On the contrary, only clearly specified events, such as “only one” or “exactly one” or “consisting of” used in the claims, are numerous or list events. This is true for the inclusion of exactly one element in. In general, the term “or” as used in this document is preceded by an exclusive term such as “one of”, “one”, “only one” or “just one”. Only if it is to be construed to indicate the exclusion of an alternative (ie, one or the other, not both). As used in the claims, “consisting essentially of” shall have its ordinary meaning as used in the field of patent law.

  The phrase “at least one” in relation to a list of one or more elements as used in the specification and claims of this application was chosen from any one or more of the element lists It should be understood to mean at least one, and does not necessarily include at least one of every element specifically listed in the element list, and does not exclude any combination of elements in the element list Should be understood to mean. This definition also allows elements other than those specifically identified in the list of elements to which the phrase “at least one” is associated, optionally present, whether or not they are associated with those particular elements. I have to. Thus, as a non-limiting example, “at least one of A and B” (in other words, at least one of A or B, in other words at least one of A and / or B), in one embodiment, B is At least one in the state of non-existing (including an element other than B as an option), including one or more as an option, A; in another embodiment, A is not present (an element other than A At least one, optionally including one or more, B; in yet another embodiment, at least one, optionally including one or more, A and at least one option Can include one or more, B (and optionally include other elements);

  Also in the claims, as in the above specification, for example, “include”, “has,” etc., “carry”, “have”, “include”, “accompany”, “hold” All transitional phrases such as “consisting of” are to be understood to mean open, ie “and including”. The transition phrases “consisting of” and “consisting essentially of” are the closed or semi-closed transition phrases, respectively, as specified in the US Patent Office Manual Patent Examination Procedure, Section 2111.03. .

Claims (23)

A method of analyzing a sample comprising a population of biological entities using at least one microfabricated device having an upper surface defining an array of microwells, wherein the plurality of microwells of the microwell array include:
(1) a target-specific nucleotide sequence for annealing to a target nucleic acid fragment of one or more biological entities of interest that may be present in the microwell, and (2) of the microwell on the microfabricated device A unique tag nucleic acid molecule that is a position-specific nucleotide sequence for identifying a position;
Loading the sample onto the microfabricated device such that at least some of the plurality of microwells each contain more than one cell of the biological entity and an amount of nutrients;
Incubating the microfabricated device under predetermined conditions;
In an individual microwell, amplifying selected genetic material of cells of the biological entity obtained from the incubation in the plurality of microwells, whereby a first in at least a subset of the plurality of microwells Acquiring amplicons;
Sequencing the collection of first amplicons collected from the subset of the plurality of microwells to obtain sequencing data; and
Identification of biological entities present in each of the plurality of microwell subsets of the at least one microfabricated device based on the sequencing data and the unique tag nucleic acid molecules contained in each of the plurality of microwells. A method consisting of the steps of earning.   The presence or absence of a relationship between at least two different types of biological entities in the population of biological entities contained in the sample based on the identification of biological entities present in each of the subsets of the plurality of microwells The method of claim 1, further comprising the step of determining.   The method of claim 2, wherein the relationship comprises a subordinate relationship, a symbiotic relationship, and a destructive relationship.   The method further comprising: determining whether the relationship is present by comparing across the plurality of microwells what kind of biological entity is present or absent in the same microwell after incubation. The method described.   Filling the microfabricated device with the sample includes, on average, including N cells of the arbitrary biological entity, where N is a number greater than or equal to two. The method of claim 2.   6. The method of claim 5, wherein N is 20 or less.   The method of claim 2, wherein the biological entity comprises a bacterium.   8. The method of claim 7, wherein the two different types of biological entities comprise two different strains, two different species, or two different genera of bacteria.   The method of claim 1, wherein the population of biological entities in the sample comprises a collection of microorganisms that are naturally present in a particular environment.   The method of claim 1, wherein the biological entity comprises a eukaryotic cell.   The method of claim 1, further comprising applying a membrane to the top surface of the microfabricated device to retain the biological entity in the plurality of microwells prior to incubation.   The method of claim 1, wherein the nutrient is pre-filled into the microwell and retained by the membrane.   The method of claim 12, wherein filling the sample comprises filling a plurality of different nutrients across the microwells of the at least one microfabricated device.   Loading the sample includes loading a plurality of different nutrients across the microwells of the at least one microfabricated device, wherein the presence or absence of a relationship between at least two types of biological entities is different. The method of claim 2 including the step of determining whether it is dependent.   The nutrient is contained in a reservoir provided on the membrane and outside the microwell, the membrane is permeable to the nutrient, and the nutrient is passed from the reservoir through the membrane to the microwell. 12. A method according to claim 11, characterized in that it is possible to move.   2. The method of claim 1, wherein the selected genetic material of the cell is genomic DNA of the cell.   In the step of transferring at least some of the cells of the biological entity obtained from the incubation to a target location, the amplification and sequencing is either a cell that is not transferred to the target location or a cell that is transferred to the target location. The method of claim 1, further comprising:   2. The method of claim 1, wherein a unique tag nucleic acid molecule in each of the plurality of microwells constitutes part of a PCR primer used for the amplification. Wherein at least one surface density of the micro-well array of microfabricated devices is at least 150 per micro well cm ^2, at least 250 microwells per cm ^2, at least 400 microwells per cm ^2, cm ² per at least 500 micro-well, cm ² At least 750 microwells per cm ^2, at least 1,000 microwells per cm ^2, at least 2,500 microwells per cm ^2, at least 5,000 microwells per cm ^2, at least 7,500 microwells per cm ^2, at least 10,000 microwells per cm ^2, at least per cm ² 50,000 microwell method of claim 1, wherein the a cm 100,000 microwells per per ² or cm ² per at least 160,000.   Each microwell of the array of microwells of the at least one microfabricated device has a diameter of about 5 μm to about 500 μm, about 10 μm to about 300 μm, or about 20 μm to about 200 μm. The method according to 1. A method for analyzing a sample comprising a population of biological entities using at least one microfabricated device having an upper surface defining an array of microwells, comprising:
Each of the plurality of microwells of the array of microwells includes (1) a target specific nucleotide sequence for annealing to a target nucleic acid fragment of one or more biological entities of interest present in the microwell; And (2) packed with a unique tag nucleic acid molecule that is a position-specific nucleotide sequence for identifying the position of the microwell on the at least microfabricated device;
Loading the sample onto the microfabricated device such that at least some of the plurality of microwells each contain more than one cell of the biological entity and an amount of nutrients. ;
Incubating the microfabricated device under predetermined conditions;
Amplifying selected genetic material of cells of a biological entity resulting from incubation in at least a subset of the plurality of microwells of the at least one microfabricated device, thereby a first unit in the plurality of microwells Obtaining a replication sequence;
Sequencing the first amplicon population collected from the subset of the plurality of microwells to obtain sequencing data; and
Based on sequencing data and unique tag nucleic acid molecules contained in each of the plurality of microwells, obtain identification of biological entities present in each of the plurality of microwell subsets of the at least one microfabricated device A method consisting of steps to do. A method for analyzing a microbiome of microorganisms collected from a particular environment using at least one microfabricated device having an upper surface defining an array of microwells, comprising:
(1) a target-specific nucleotide sequence for annealing to a target nucleic acid fragment of one or more biological entities of interest that may be present in the microwell, and (2) for identifying the location of the microwell Comprising a position-specific nucleotide sequence;
A sample prepared from the microbiome is loaded onto the at least one microfabricated device such that the plurality of microwells each averages 2 to 20 microbiome cells of any microorganism and a certain amount of nutrients Including:
Incubating the microfabricated device under predetermined conditions;
Amplifying selected genetic material of the microorganism resulting from the incubation in at least a subset of the plurality of microwells in the at least one microfabricated device, thereby providing a first amplicon in the plurality of microwells Step to acquire;
Sequencing aggregates of a first amplicon collected from the subset of the plurality of microwells to obtain sequencing data; and
Based on the sequencing data and a unique tag nucleic acid molecule contained in each of the plurality of microwells, obtaining an identification of microorganisms present in each of the plurality of microwell subsets of the at least one microfabricated device;
Determining the presence or absence of a relationship between at least two different types of microorganisms in the microbiome based on the identification of microorganisms present in the plurality of microwells. A method for analyzing the relationship between microbiome microorganisms collected from a particular environment, comprising:
In the step of dividing the microbiome microorganisms into a plurality of portions, each comprising an average of 2-20 cells of microbiome microorganisms;
Incubating each portion of the plurality of portions of the microorganism in separate compartments in the presence of the nutrients under predetermined conditions;
After incubation, identifying microorganisms present in each of at least a subset of the isolated compartments; and
Determining the presence or absence of a relationship between at least two different types of microorganisms in the microbiome based on a comparison across a subset of compartments whether the identified microorganism types are present or absent in the same microwell A method consisting of:

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