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US 2020/0347448 A1Published application
Functional metagenomics
AssigneeWild Biotech
InventorsIdo Bachelet, Moran Avraham Kopel, Liron Anna Bassali, Neta Raab
Life of this patent
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Abstract
The present disclosure describes methods for identifying a microbe that modulates an activity of a target cell, a target virus, or an enzymatic reaction of a target substrate.
Claims99
A method for identifying a microbe that modulates an activity of a target cell or virus, the method comprising:
- (a) forming a test sample comprising a plurality of target cells or viruses, and a mixed microbial population;
- (b) forming droplets from the test sample such that each droplet contains no more than one microbe from the mixed microbial population and a predetermined number of target cells or viruses;
- (c) incubating the droplets under conditions and for a period of time sufficient to detect a predetermined amount of activity of the target cells or viruses in the absence of a microbe;
- (d) isolating a droplet in which the target cell or virus had a different amount of activity than the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the isolated droplet to identify the microbe.
3 more elements
A method for identifying a microbe that inhibits replication or survival of a microbial cell, the method comprising:
- (a) forming a test sample comprising a plurality of microbial cells, a mixed microbial population, a set of barcoded primers comprising a universal priming sequence, a unique barcode and a universal microbial sequence, and a plurality of universal primers specific for the universal priming sequence;
- (b) forming droplets from the test sample such that each droplet contains no more than one microbe from the mixed microbial population, a predetermined number of microbial cells, no more than 1 barcoded primer, and a plurality of universal primers;
- (c) incubating the droplets under conditions and for a period of time sufficient to detect a predetermined amount of replication or survival of the microbial cells in the absence of a microbe;
- (d) performing an emulsion PCR reaction to generate barcode-labeled amplification product, wherein each unique barcode is associated with a single droplet in which the amplification took place;
- (e) performing next-generation sequencing analysis of the amplification products to identify microbial sequences from droplets in which the microbial cell had a different amount of replication or survival from the predetermined amount.
2 more elements
A method for identifying a microbe that modulates an activity of a target cell or virus, the method comprising:
- (a) forming a test sample comprising a plurality of target cells or viruses, and a mixed microbial population;
- (b) distributing the test sample into microwells such that each microwell contains no more than one microbe from the mixed microbial population and a predetermined number of target cells or viruses;
- (c) incubating the microwells under conditions and for a period of time sufficient to detect a predetermined amount of activity of the target cells or viruses in the absence of a microbe;
- (d) detecting a microwell in which the target cell or virus had a different amount of activity from the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the detected microwell to identify the microbe.
3 more elements
A method for identifying a microbe that inhibits replication or survival of a microbial cell, the method comprising:
- (a) forming a test sample comprising a plurality of microbial cells, a mixed microbial population, a set of barcoded primers comprising a universal priming sequence, a unique barcode and a universal microbial sequence, and a plurality of universal primers specific for the universal priming sequence;
- (b) distributing the test sample into microwells such that each microwell contains no more than one microbe from the mixed microbial population, a predetermined number of microbial cells, no more than 1 barcoded primer, and a plurality of universal primers;
- (c) incubating the microwells under conditions and for a period of time sufficient to detect a predetermined amount of replication or survival of the microbial cells in the absence of a microbe;
- (d) performing an emulsion PCR reaction to generate barcode-labeled amplification product, wherein each unique barcode is associated with a single microwell in which the amplification took place;
- (e) performing next-generation sequencing analysis of the amplification products to identify microbial sequences from droplets in which the microbial cell had a different amount of replication or survival from the predetermined amount.
2 more elements
A method for identifying a microbe that modulates an enzymatic reaction of a target substrate, the method comprising:
- (a) forming a test sample comprising a plurality of target substrates and a mixed microbial population;
- (b) forming droplets from the test sample such that each droplet contains no more than one microbe from the mixed microbial population and a predetermined amount of target substrates;
- (c) incubating the droplets under conditions and for a period of time sufficient to detect a predetermined amount of enzymatic reaction of the target substrate in the absence of a microbe;
- (d) isolating a droplet in which the target substrate underwent a different amount of enzymatic reaction from the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the isolated droplet to identify the microbe.
3 more elements
A method for identifying a microbe that modulates an enzymatic reaction of a target substrate, the method comprising:
- (a) forming a test sample comprising a plurality of target substrates and a mixed microbial population;
- (b) distributing the test sample into microwells such that each microwell contains no more than one microbe from the mixed microbial population and a predetermined amount of target substrates;
- (c) incubating the microwells under conditions and for a period of time sufficient to detect a predetermined amount of enzymatic reaction of the target substrate in the absence of a microbe;
- (d) detecting a microwell in which the target substrate underwent a different amount of enzymatic reaction from the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the detected microwell to identify the microbe.
3 more elements
A method for identifying a microbe that protects a host cell against viral infection, the method comprising:
- (a) forming a test sample comprising a plurality of host cells, a mixed microbial population and a plurality of target viruses;
- (b) forming droplets from the test sample such that each droplet contains no more than one microbe from the mixed microbial population, a predetermined number of target viruses, and a predetermined number of host cells;
- (c) incubating the droplets under conditions and for a period of time sufficient to detect a predetermined amount of activity of the host cells in the presence of the target viruses but absence of a microbe;
- (d) isolating a droplet in which the host cell had a different amount of activity than the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the isolated droplet to identify the microbe.
3 more elements
A method for identifying a microbe that protects a host cell against viral infection, the method comprising:
- (a) forming a test sample comprising a plurality of host cells, a plurality of target viruses, and a mixed microbial population;
- (b) distributing the test sample into microwells such that each microwell contains no more than one microbe from the mixed microbial population, a predetermined number of target viruses, and a predetermined number of host cells;
- (c) incubating the microwells under conditions and for a period of time sufficient to detect a predetermined amount of activity of the host cells in the presence of the target viruses but absence of a microbe;
- (d) detecting a microwell in which the host cell had a different amount of activity from the predetermined amount;
- and
- (e) sequencing DNA from the microbe in the detected microwell to identify the microbe.
3 more elements
Dependencies are inferred from the claim text.