Microbial Genome Sequencing via Barcoded WGA
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Solution Overview
Problem
Current methods for identifying and sequencing unknown microbes, such as bacteria and archaea, are impractical due to the need for culturing and the inefficiency of shotgun sequencing, which often results in wasted data from dominant strains, making it difficult to isolate and sequence unculturable microbes.
Innovation Solution
The method involves selecting biological samples for DNA sequencing to identify Unique Sequence Identifiers (USIDs), diluting samples for whole genome amplification, and sequencing these USIDs to detect and isolate unknown microbes, allowing for their genomic sequencing without the need for culturing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If shotgun sequencing is used on complex microbiome samples, then sequence data is obtained, but most data is wasted from dominant strains making unknown microbes difficult to detect
Solution Approach 1:
The method extracts and isolates specific target DNA sequences (16S rRNA genes) from complex microbiome samples using PCR amplification with universal primers. This extraction approach separates the signal of interest (unknown microbes) from the overwhelming background noise of dominant strains, enabling focused sequencing that eliminates wasted data from abundant taxa while maintaining high detection sensitivity for rare unknown microbes
Solution Approach 2:
The patent applies local quality by targeting specific hypervariable regions (V1-V9) of the 16S rRNA gene that provide species-specific identification capability. By focusing sequencing efforts on these locally optimized target regions rather than whole genomes, the method achieves high discriminatory power for unknown microbe detection while minimizing data waste from dominant strains that would otherwise overwhelm the sequencing capacity
2Quantity of substance
If culturing methods are used to obtain sufficient material for sequencing, then enough DNA is obtained, but unculturable microbes cannot be sequenced
Solution Approach 1:
The patent uses PCR amplification as an intermediary technique to bridge the gap between limited starting material and sufficient sequencing input. Universal primers targeting conserved 16S rRNA regions serve as mediators that can amplify DNA from any bacterial or archaeal cell, whether culturable or not. This intermediary approach eliminates the need for culturing while still generating adequate DNA quantities for whole genome sequencing of unknown microbes
Solution Approach 2:
The method replaces the mechanical/biological process of culturing with an in vitro enzymatic amplification system (PCR). Instead of relying on microbial growth in culture media, the patent uses polymerase chain reaction to exponentially amplify target DNA sequences directly from environmental samples, thereby enabling sequencing of unculturable microbes while maintaining versatility across diverse microbial taxa
3Measurement precision
If whole genome amplification is performed on diluted samples, then single microbes can be sequenced, but the process must be high throughput to be practical
Solution Approach 1:
The patent merges multiple individual microbe sequencing reactions into a single high-throughput sequencing run by pooling barcoded amplicons from numerous samples. Each sample receives a unique molecular barcode during PCR, allowing thousands of individual microbe genomes to be multiplexed and sequenced simultaneously on next-generation sequencing platforms. This merging approach maintains the precision of single-microbe sequencing while achieving the productivity needed for practical application across complex microbiome studies
4Loss of time
If barcoding and high throughput sequencing are used, then costs and time are reduced, but the method must maintain accuracy in identifying unknown microbes
Solution Approach 1:
The patent performs preliminary action by adding unique molecular barcodes to DNA amplicons from each sample during the PCR amplification step, before sequencing occurs. This pre-tagging allows subsequent high-throughput pooling and sequencing of hundreds of samples without losing sample identity. The barcode assignment happens in advance, enabling rapid parallel processing while maintaining the ability to accurately attribute sequencing reads to their source unknown microbes, thus reducing time without sacrificing identification accuracy
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
This approach enables rapid and efficient identification, quantification, and sequencing of unknown microbes in mixed samples, including those that are unculturable, by using phi29 DNA polymerase for whole genome amplification and barcoding for high-throughput sequencing, reducing costs and time while minimizing bias.
Implementation Method 1
amplifying whole genome DNA from the single microbes in step (b)
Implementation Method 2
conducting DNA sequencing on the whole genome amplified DNA of the unknown microbe identified in step (d), and assembling the DNA sequences into the genome of the unknown microbe
Data Source
AI summary
Disclosed are methods for screening biological samples for the presence unknown microbes, such as bacteria and archaea or unknown eukaryotes using rRNA gene sequences or other highly conserved genetic regions, across multiple biological samples using a unique sequence tag (barcode) corresponding to the sample. The screening process tracks the unknown microbe or eukaryote in a diluted sample where the DNA has been prepared using whole genome amplification. The whole genome of the unknown microbe or eukaryote is then sequenced and assembled.