High resolution systems, kits, apparatus, and methods for high throughput microbiology applications
Inventors
Blainey, Paul C. • Seely, Michael W. • Stocker, Roman • Zengler, Karsten • Conradson, Scott • Christey, Peter • Hallock, Alexander
Assignees
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Publication Number
US-10900073-B2
Publication Date
2021-01-26
Expiration Date
Abstract
A microfabricated device defining a high density array of microwells is described for cultivating cells from a sample. A series of unique tags may be disposed in the microwells to identify one or more species of cells and locate the particular microwells in which each species was cultivated. A unique tag may be a nucleic acid molecule including a target-specific nucleotide sequence for annealing to a target nucleic acid fragment and a location-specific nucleotide sequence predetermined to identify one or more microwells. The device may be incubated to grow a plurality of cells, which may be split into an analysis portion and a reserve portion. High throughput methods are described for cultivating, screening, and determining a relative and/or absolute abundance of cells from a sample.
Core Innovation
The invention relates to microfabricated devices that define a high density array of microwells for cultivating at least one cell from a sample. At least one microwell includes at least one unique tag comprising a nucleic acid molecule, where the nucleic acid molecule includes a target-specific nucleotide sequence for annealing to a target nucleic acid fragment and a location-specific nucleotide sequence for identifying the microwell. Cells are incubated to grow a plurality of cells in the tagged microwells, enabling identification of the spatial location where particular cells are cultivated.
After incubation, the plurality of cells in a microwell is split into a first portion and a second portion, with the at least one unique tag remaining with at least the first portion. The first portion is analyzed to determine the at least one species of interest and to identify the microwell in which the at least one species of interest is cultivated based on the at least one unique tag. Based on identifying the microwell, the invention locates the at least one species of interest within the second portion, thereby maintaining locational information for cultivated species.
The invention further supports abundance determination by cultivating cells in a microfabricated device with a high density array of microwells where sample loading provides that no more than one cell is disposed in each microwell for a preponderance of the microwells. Determining based on the at least one unique tag a first number of microwells comprising at least one cell and a second number of microwells comprising the at least one species of interest enables relative abundance of the at least one species of interest in the sample. In related embodiments, biological entities are cultivated in a high density array of experimental units and uniquely tagged to identify spatial information, where the unique tag includes a first binding molecule with target-specific and location-specific components.
Claims Coverage
The partial content includes three independent claims. Across these claims, the inventive coverage focuses on microfabricated high density arrays with uniquely tagged spatial identification, maintaining tag association during splitting to enable location of analyzed species, and using tag-based counting to determine relative abundance.
Locational indexing of cultivated species via nucleic-acid unique tags
Obtaining a microfabricated device defining a high density array of microwells for cultivating at least one cell from a sample, where at least one microwell comprises at least one unique tag having a nucleic acid molecule with a target-specific nucleotide sequence for annealing to a target nucleic acid fragment and a location-specific nucleotide sequence for identifying the microwell; incubating to grow a plurality of cells in the microwells; splitting the plurality of cells in the microwell into a first portion and a second portion while the unique tag remains with at least the first portion; analyzing the first portion to determine the species of interest and identify the microwell based on the unique tag; and locating the species of interest within the second portion based on identifying the microwell.
Relative abundance determination using uniquely tagged microwells with single-occupancy loading
Obtaining a microfabricated device defining a high density array of microwells for cultivating at least one cell from a sample, where each microwell comprises at least one unique tag including a nucleic acid molecule with a target-specific nucleotide sequence and a location-specific nucleotide sequence predetermined to identify the microwell; preparing and loading the sample such that no more than one cell is disposed in each microwell of a preponderance of the microwells; and determining based on the unique tag a first number of microwells comprising at least one cell and a second number of microwells comprising the species of interest, and determining a relative abundance of the species of interest based on the first number and the second number.
Spatial information identification for cultivated biological entities using target-specific and location-specific binding molecules
Obtaining a microfabricated device defining a high density array of experimental units for cultivating biological entities from a sample, where at least one experimental unit comprises at least one unique tag to identify spatial information relating to the experimental unit, the unique tag comprising a first binding molecule including a target-specific component for binding to a biomolecule present in a type of interest and a location-specific component for identifying the spatial information; wherein the biological entity is at least one of an organism, a cell, a cell component, a cell product, and a virus, and the first binding molecule is at least one of a protein, a nucleic acid, a carbohydrate, a lipid, and a drug; incubating to grow a plurality of biological entities from the biological entity in the experimental unit; and analyzing the plurality of biological entities to determine the type of interest and identify the spatial information relating to the experimental unit where the type of interest is cultivated based on the unique tag.
Across the independent claims, the document covers high density microfabricated arrays associated with uniquely tagged spatial information: nucleic-acid unique tags enable tag-based microwell identification of cultivated species and locational retrieval after splitting; uniquely tagged microwells with controlled single-occupancy enable tag-based counting for relative abundance; and target-specific plus location-specific binding molecules enable spatial information identification for a broad set of biological entities.
Stated Advantages
Enable locational identification of a species of interest within a second portion of cells based on tag-based identification of the microwell where the species is cultivated.
Determine relative abundance of at least one species of interest in a sample based on tag-based counts of microwells comprising at least one cell and microwells comprising the species of interest.
Determine the type of interest of biological entities and identify spatial information relating to the experimental unit where the type of interest is cultivated based on a unique tag.
Documented Applications
Cultivating microbial and other biological entities in microfabricated devices with high density microwell arrays and uniquely barcoded/location-specific tags to allow locational indexing of species in individual wells after incubation.
Determining relative abundance of at least one species of interest in a sample using uniquely tagged microwells with no more than one cell disposed in each microwell for a preponderance of microwells.
Cultivating biological entities in a high density array of experimental units and determining both the type of interest and spatial information relating to where cultivation occurred based on a unique tag.
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