High resolution systems, kits, apparatus, and methods for bacterial community relationship determination and other high throughput microbiology applications
Inventors
Christey, Peter • Hallock, Alexander
Assignees
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Publication Number
US-10788452-B2
Publication Date
2020-09-29
Expiration Date
Abstract
A method is provided for analyzing a sample including a population of biological entities using at least one microfabricated device. A plurality of the microwells on the microfabricated device are each uniquely indexed, and loaded with a sample such that at least some microwells each include more than one cell of a biological entity. The microfabricated device was incubated at predetermined conditions, and a selected genetic material of the cells of the biological entities obtained from the incubation is amplified to obtaining amplicons. An aggregate of the amplicons are sequenced obtain sequencing data, based on which and the indexing of the microwells, an identification of the biological entities present in each of the plurality of microwells is obtained. Such identification can then be used to determine a relationship between different types of biological entities in the sample.
Core Innovation
The invention provides a method of analyzing a sample including a population of biological entities using at least one microfabricated device having a top surface defining an array of microwells. A plurality of the microwells each comprise a unique tag nucleic acid molecule that includes a target-specific nucleotide sequence and a location-specific nucleotide sequence, enabling identification of both a target nucleic acid fragment and the location of the microwell on the device.
The method loads a sample onto the microfabricated device so that at least some microwells each include more than one cell of a biological entity and an amount of a nutrient, followed by incubating the device at predetermined conditions. Selected genetic material from cells obtained from the incubation is amplified in individual microwells to obtain first amplicons in at least a subset of the microwells, and an aggregate of the first amplicons collected from the subset is sequenced to obtain sequencing data.
Based on the sequencing data and the unique tag nucleic molecule included in each microwell, the method obtains an identification of biological entities present in each microwell of the subset. Based on the identification of biological entities present in each microwell, the method determines a presence or absence of a relationship regarding co-inhabitance between at least two different types of cells of biological entities in the population contained in the sample.
Claims Coverage
Independent claims clm-00001, clm-00020, clm-00021, and clm-00022 each cover a microfabricated microwell-based workflow with unique tag nucleic acids that link target identification to microwell location, and a determination of presence or absence of co-inhabitance relationships across microwells or compartments. Across the independent claims, the core inventive features include four features: unique target-specific and location-specific tagging per microwell, microwell partitioning with nutrient and incubation at predetermined conditions, microwell-level amplification and aggregate sequencing to generate sequencing data, and determination of co-inhabitance relationships from per-microwell identifications.
Microfabricated device with unique tag nucleic acid per microwell
A microfabricated device having a top surface defining an array of microwells, wherein a plurality of the microwells each comprise a unique tag nucleic acid molecule including a target-specific nucleotide sequence and a location-specific nucleotide sequence for identifying the location of the microwell on the microfabricated device.
Microwell loading with nutrients and multiple cells per microwell
Loading a sample onto the microfabricated device such that at least some microwells each include more than one cell of a biological entity as well as an amount of a nutrient.
Microwell amplification with first amplicons and aggregate sequencing
Amplifying, in individual microwells, a selected genetic material of the cells obtained from the incubation to obtain first amplicons in at least a subset of the plurality of microwells, and sequencing an aggregate of the first amplicons collected from the subset to obtain sequencing data.
Per-microwell identification using sequencing data and unique tag
Based on the sequencing data and the unique tag nucleic molecule included in each of the plurality of the microwells, obtaining an identification of the biological entities present in each of the subset of the plurality of microwells of the at least one microfabricated device.
Co-inhabitation relationship determination based on per-microwell identifications
Based on the identification of the biological entities present in each of the subset of the plurality of microwells, determining a presence or absence of a relationship regarding co-inhabitance between at least two different types of cells of biological entities in the population contained in the sample.
Microbiome analysis using unique tagged microwells
Loading a sample prepared from the microbiome onto the at least one microfabricated device such that the plurality of the microwells each include, on average, 2 to 20 cells of any microorganism of the microbiome as well as an amount of a nutrient, followed by incubating at predetermined conditions, amplifying selected genetic material in at least a subset of the microwells, sequencing an aggregate of the first amplicons to obtain sequencing data, and obtaining an identification of microorganisms present in each microwell.
Microbiome co-inhabitance relationship determination across microorganisms
Based on the identification of the microorganisms present in the plurality of microwells, determining a presence or absence of a relationship regarding co-inhabitance between at least two different types of microorganisms in the microbiome.
Partitioning microbiome microorganisms into portions and incubating
Partitioning microorganisms of the microbiome into a plurality of portions each including, on average, 2 to 20 cells of any microorganism of the microbiome; incubating each portion in a separate compartment at predetermined conditions in the presence of a nutrient; and identifying the microorganisms present in each of at least a subset of the isolated compartments after incubation.
Co-inhabitance relationship determination by comparison across compartments
Determining a presence or absence of a relationship regarding co-inhabitance between at least two different types of the microorganisms in the microbiome based on a comparison across the subset of compartments which types of microorganisms as identified are present or not present in the same microwells.
Across the independent claims, the shared claim coverage centers on a microfabricated device with an array of microwells where each microwell includes a unique tag nucleic acid with a target-specific sequence and a location-specific sequence, enabling per-microwell identifications from sequencing data after incubation and amplification. The claims then determine presence or absence of co-inhabitance relationships using those per-microwell identifications, with clm-00021 specifying microbiome analysis and clm-00022 providing an alternative compartment/portion partitioning comparison approach.
Stated Advantages
Allows determining a presence or absence of a relationship regarding co-inhabitance between at least two different types of cells of biological entities in the population contained in the sample.
Enables obtaining an identification of biological entities present in each subset of microwells based on sequencing data and the unique tag nucleic molecule.
Enables obtaining an identification of microorganisms present in each subset of microwells for microbiome samples.
Enables determining a presence or absence of a relationship regarding co-inhabitance between at least two different types of microorganisms in a microbiome.
Documented Applications
Analyzing a sample containing a population of biological entities to determine co-inhabitance relationships among biological entity types.
Analyzing a microbiome of microorganisms collected from a specific environment to obtain microorganism identifications and determine co-inhabitance relationships.
Analyzing a relationship among microorganisms of a microbiome collected from a specific environment by comparing identifications across isolated compartments/portions.
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