PCR-Activated Sorting for Rare Microbe Genome Recovery
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Solution Overview
Problem
Current methods for studying microbial communities, particularly uncultivable microbes, face challenges due to the complexity and diversity of nucleic acids, leading to difficulties in sequencing and detecting rare microbes, as well as the limitations of probe hybridization capture and fluorescence-activated cell sorting (FACS) techniques.
Innovation Solution
The development of PCR-Activated Sorting (PAS) methods, which involve encapsulating samples in microdroplets, performing PCR amplification, and using fluorescent assays to label and sort target nucleic acids, allowing for the enrichment of target sequences while discarding off-target DNA, enabling the recovery of whole microbial genomes without the need for extensive probe design or chemical fixation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If shotgun sequencing is used to study uncultivable microbes, then all nucleic acids in the sample can be sequenced, but the analysis becomes convoluted and rare microbes are swamped out by off-target sequences
Solution Approach 1:
The patent segments the sequencing process by first enriching for target microbial cells using FISH-FACS sorting before sequencing. This divides the heterogeneous nucleic acid mixture into distinct target and off-target fractions, allowing rare microbes to be studied without being swamped by abundant sequences from other organisms.
Solution Approach 2:
The patent performs preliminary enrichment of target microbial cells through FISH-FACS sorting before sequencing. By pre-concentrating the rare microbes of interest and removing abundant off-target sequences beforehand, the subsequent sequencing yields much higher information content about the rare microbes without the convolution problem.
2Measurement precision
If probe hybridization capture is used to enrich target genomes, then specific microbial sequences can be recovered, but hundreds or thousands of overlapping probes are required and fragments captured are limited to those near targeted sequences
Solution Approach 1:
The patent extracts and enriches for intact target microbial cells from the heterogeneous sample using FISH-FACS sorting before genome recovery. This approach takes out the entire genome of interest along with the cell, avoiding the need for numerous capture probes and ensuring complete genome recovery rather than just fragments near probe targets.
Solution Approach 2:
The patent uses fluorescently-labeled probes to identify and sort target cells, creating a detectable copy signal that guides the sorting process. This copying approach allows for precise identification of target cells without requiring physical capture probes for every genomic region, simplifying the overall process.
3Ease of manufacture
If FISH-FACS is used to label and sort target cells, then whole genomes can be recovered without probe design, but chemical fixation and permeabilization may modify DNA and introduce sequencing bias
Solution Approach 1:
The patent optimizes the fixation and permeabilization parameters to minimize DNA modification while maintaining cell integrity for sorting. By carefully controlling the duration and conditions of chemical treatment, the method preserves genetic material integrity sufficiently for downstream sequencing applications.
Solution Approach 2:
The patent uses fluorescently-labeled probes as intermediaries to bind to target microbial nucleic acids, enabling specific identification and sorting without requiring direct manipulation of the genetic material itself. This intermediary approach allows for precise sorting while minimizing harmful effects on the DNA.
4Measurement precision
If horizontal gene transfer is detected using traditional methods, then known sequences can be identified, but unpredictable transferred sequences are missed because probes cannot be designed for unknown elements
Solution Approach 1:
The patent employs a dynamic approach by using 16S rRNA gene targeting for initial enrichment followed by whole genome sequencing. This allows the method to adapt to and detect any horizontal gene transfer events, whether known or unknown, because the initial enrichment is based on a conserved marker while the final sequencing captures the entire genomic content including unpredictable transferred elements.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
PAS effectively enriches for target nucleic acids, improving the detection of rare microbes and overcoming the limitations of existing techniques by providing a cultivation-free, high-throughput method for sequencing diverse microbial communities, preserving the integrity of genetic material and reducing sequencing bias.
Implementation Method 1
The microdroplets are subjected to PCR amplification conditions, such that if a microdroplet contains a nucleic acid corresponding to a target of interest
Implementation Method 2
detecting the presence or absence of the PCR amplification products by detection of the detection component
Implementation Method 3
encapsulating an aqueous sample, which may include a heterogeneous population of cells, viruses, and/or nucleic acids, in a plurality of microdroplets, wherein each microdroplet includes an aqueous phase fluid in an immiscible phase carrier fluid
Data Source
AI summary
The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.