16S rRNA Quantification via Segmented qPCR and Sequencing
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Solution Overview
Problem
Current methods for nucleic acid quantification in microbial communities face challenges in achieving accurate and robust quantification across a wide dynamic range with minimal interference from contaminant nucleic acids and biases, especially in complex mixtures.
Innovation Solution
A method and system for absolute quantification of 16S rRNA and prokaryotes using specific primers that amplify and sequence a 16S rRNA recognition segment, allowing for the calculation of absolute abundance by multiplying relative abundance by absolute abundance of sample 16S rRNAs, which reduces non-specific mammalian mitochondrial DNA amplification and provides precise quantification even in high host DNA backgrounds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid quantification methods are used in complex microbial communities, then quantification can be performed, but accuracy and robustness deteriorate due to interference from contaminant nucleic acids and biases in complex mixtures
Solution Approach 1:
The method segments the quantification process into two independent components: (1) absolute quantification of total 16S rRNA copies using qPCR, and (2) relative abundance determination of specific taxa using amplicon sequencing. This segmentation allows each component to be optimized independently, with qPCR providing accurate total abundance measurements unaffected by sequencing biases, while sequencing provides taxonomic composition. The combination yields precise absolute abundance data for specific taxa without interference from contaminant nucleic acids.
Solution Approach 2:
The patent uses the 16S rRNA gene as an intermediary marker that is universally present in prokaryotes but absent in eukaryotic host cells. By targeting this specific genetic marker with taxon-specific primers, the method indirectly quantifies microbial abundance without directly interacting with contaminant host DNA. The intermediary approach allows selective amplification of microbial sequences even in samples with high host DNA background, effectively filtering out contaminant interference.
2Adaptability or versatility
If broad microbial diversity is targeted for quantification, then comprehensive community profiling is achieved, but quantification robustness deteriorates due to increased complexity and potential biases in complex nucleic acid mixtures
Solution Approach 1:
The method divides the complex quantification task into manageable segments: total abundance measurement via qPCR and taxonomic distribution via sequencing of the 16S rRNA gene. This segmentation enables the system to handle broad microbial diversity through the versatile sequencing component while maintaining robustness through the reliable qPCR-based absolute quantification component. Each segment can be independently optimized for its specific function.
Solution Approach 2:
The 16S rRNA gene serves as a universal marker that enables simultaneous quantification of diverse prokaryotic taxa through a single molecular target. The conserved regions of the 16S gene allow design of universal primers that can amplify sequences from a wide range of bacterial and archaeal species, providing multi-functional capability for comprehensive community profiling while maintaining a standardized approach that ensures robustness across different samples and taxa.
3Adaptability or versatility
If wide dynamic range quantification is achieved, then samples with varying microbial loads can be analyzed, but measurement precision deteriorates due to increased difficulty in detecting and measuring across multiple orders of magnitude
Solution Approach 1:
The method employs periodic action through the use of standard curves in qPCR, where known concentrations of template DNA are amplified in a series of dilutions to establish a reference relationship between cycle threshold and log concentration. This periodic calibration approach enables accurate quantification across a wide dynamic range by providing reference points at multiple concentration levels, allowing precise measurement of unknown samples regardless of their microbial load magnitude.
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
Enables accurate and precise quantification of microbial communities with a wide dynamic range, reducing the impact of contaminants and biases, and allowing for the analysis of samples with varying microbial loads and high host DNA concentrations.
Implementation Method 1
amplifying the 16S rRNA recognition segment in nucleic acids extracted from the sample with primers comprising a target primer sequence specific for the target 16S rRNA conserved regions
Implementation Method 2
sequencing the 16S rRNA recognition segment with primers comprising the target primer sequence specific for the target 16S rRNAs conserved region to detect a relative abundance of the target 16S rRNA
Data Source
AI summary
Provided herein are methods and systems for absolute quantification of a target 16S rRNA and/or of a target prokaryotic taxon, based on amplifying and sequencing a same 16S rRNA recognition segment in which target 16S rRNA conserved regions flank 16S rRNA variable regions, conserved and variable among a plurality of sample 16S rRNAs and/or of a sample prokaryotic taxon of higher taxonomic rank with respect to the target taxon. In the methods and systems, absolute abundance of the a plurality of sample 16S rRNAs and/or of the sample prokaryotic taxon detected by the amplifying, is multiplied by the relative abundance of the target 16S rRNA and/or of a target prokaryotic taxon detected by the sequencing to provide the absolute quantification in accordance with method and systems of the disclosure.