Systems and method for electrophoretic fractionation of the microbiome
Inventors
Dovichi, Norman • Jaskowski Huge, Bonnie • Champion, Matthew
Assignees
Publication Number
US-11746388-B2
Publication Date
2023-09-05
Expiration Date
2038-03-07
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Abstract
A system and methods of characterizing a population of a virus, bacteriophage, or other microbes in a microbiome that includes the steps of separating a sample of microbiota into more than one fraction by continuous capillary zone electrophoresis based on the physiochemical properties of the microbes in the microbiota using a constant voltage applied to the sample during the continuous zone electrophoresis. At least one separated fraction includes an intact virus, bacteriophage, or other microbe that may be visualized and/or directly sequenced to characterize the population of the virus, bacteriophage, or other microbe in the microbiome.
Core Innovation
The invention provides a system and method for characterizing a population of viruses, bacteriophages, or other microbes in a microbiome by utilizing continuous capillary zone electrophoresis to separate a sample of microbiota into multiple fractions based on the physiochemical properties of the microbes. This is achieved by applying a constant voltage to the sample during electrophoresis, enabling fractionation in which at least one fraction contains an intact virus, bacteriophage, or other microbe. The separated fractions may then be visualized or directly sequenced to analyze and characterize the microbial population in detail.
Current methods for characterizing environmental microbiomes are limited by the dominance of highly abundant species in sequencing data, making the identification of rare species inefficient and challenging. Traditional approaches such as next-generation sequencing require significant oversampling to detect low abundance species, and only a small subset of microbiota can be cultured, leaving most species undetectable by classic microbiological techniques. Marker-based experiments and closed-reference analyses also obscure diversity and miss sequences absent from reference databases, further impairing comprehensive analysis.
The disclosed device includes a separation capillary connected to an injection block for the sample, a power source supplying voltage, a dispensing valve with a deposition buffer container, a nozzle and collector connected through a tee fitting, and a nucleic acid sequencer interfaced with the fraction collector. This configuration allows for the isolation of microbial fractions that can be further analyzed, minimizing the need for oversampling and eliminating the necessity for culturing microbes in each fraction. Methods involve inserting the sample, separating by electrophoresis, collecting fractions, optionally amplifying nucleic acid, and sequencing, enabling efficient identification of both common and rare species in complex microbiome samples.
Claims Coverage
There is one independent claim covering the main inventive features of the system and method for analyzing a virus or bacteriophage microbiome using capillary zone electrophoresis and downstream nucleic acid sequencing.
System for capillary zone electrophoresis-based microbiome fractionation and sequencing
A device is provided comprising: - A separation capillary with proximal and distal ends, where the proximal end is connected to an injection block configured for a virus or bacteriophage microbiota sample. - A power source configured to supply a voltage across the separation capillary. - A background buffer with one or more electrolytes and a pH of about 3 to about 11. - A dispensing valve in fluidic connection to a deposition buffer container. - A nozzle in fluidic connection to the dispensing valve and distal end of the capillary through a tee fitting. - A fraction collector with a collector plate on a movable stage positioned below the nozzle. - A nucleic acid sequencer interfaced with the fraction collector. - The sample is separated within the capillary, deposited as isolated fractions, and subsequently analyzed by nucleic acid sequencing.
Method for analyzing virus or bacteriophage microbiome by electrophoretic separation, fraction collection, and sequencing
The method includes: 1. Inserting a sample of virus or bacteriophage microbiota into the injection block. 2. Applying a constant voltage to the separation capillary to achieve separation into fractions. 3. Pressurizing the deposition buffer container containing deposition buffer. 4. Opening the dispensing valve to facilitate collection. 5. Collecting fractions of purified, intact virus or bacteriophage microbiota separated from the mixture. 6. Optionally amplifying the purified material. 7. Visualizing the fractionated virus or bacteriophage using electron microscopy. 8. Sequencing nucleic acid from the fractionated material using the interfaced sequencer. Analysis is performed on the microbiome within each fraction by nucleic acid sequencing.
The independent claim covers a device and method utilizing capillary zone electrophoresis for microbiome sample fractionation, enabling isolation and downstream genetic or visualization analysis of viruses, bacteriophages, or other microbes within a complex microbiome sample.
Stated Advantages
Enables identification and analysis of rare microbial species in complex microbiome samples by separating them from abundant species.
Reduces or eliminates the need for oversampling in sequencing experiments, improving efficiency and data quality.
Permits direct genetic sequencing of microbes without requiring prior culturing, facilitating analysis of non-culturable organisms.
Provides a highly automated and reproducible system for sample separation and fraction collection.
Enhances the number of operational taxonomic units (OTUs) identified compared to unfractionated samples.
Allows for downstream genetic or biochemical analysis, including visualization via electron microscopy.
Requires no prior knowledge or modification of the microorganisms for downstream analysis.
Documented Applications
Characterization and analysis of virus, bacteriophage, and microbial populations in environmental and clinical microbiome samples.
Isolation and identification of rare microbial species and strains from complex mixtures for genetic sequencing.
Visualization of separated viruses or bacteriophages using electron microscopy.
Research in areas such as environmental monitoring, wastewater treatment, and studying microbiota from plants, animals, or human-derived samples.
Preparation of purified microbial fractions for further genetic or biochemical analysis, including next-generation sequencing of 16S rRNA or whole-genome sequencing.
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