Multiplex Tag Sequencing for High-Throughput Ecogenomics
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current sequencing technologies face limitations in speed, accuracy, and cost, particularly in clinical and environmental applications that require processing multiple samples, leading to reduced throughput and increased costs.
Innovation Solution
The development of multiplex methods for determining polynucleotide sequences in multiple samples using tag sequences, which allow for the identification and quantification of variants without the need for Southern blot transfer or size-separating primer extension products and electrophoresis, enabling simultaneous sequencing of multiple samples in a mixture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional Sanger sequencing methods are used, then sequence determination accuracy is maintained, but processing speed and throughput are limited
Solution Approach 1:
The invention segments the sequencing process by attaching unique molecular identifiers (UMIs) to individual polynucleotide molecules, allowing parallel processing of multiple samples. Each molecule is tagged and processed independently, enabling high-throughput sequencing without compromising accuracy through statistical analysis of multiple tagged molecules.
Solution Approach 2:
The invention performs preliminary tagging of polynucleotide molecules with unique identifiers before sequencing. This preliminary action allows for subsequent high-throughput processing while maintaining the ability to track and analyze individual molecules, resolving the contradiction between speed and accuracy.
2Productivity
If multiple samples are processed simultaneously, then productivity increases, but measurement precision and accuracy may deteriorate
Solution Approach 1:
Each polynucleotide molecule is segmented with a unique molecular identifier tag, allowing individual tracking even when multiple samples are processed simultaneously. This segmentation enables the system to maintain measurement precision for each molecule while achieving high overall productivity through parallel processing.
Solution Approach 2:
The invention creates multiple copies of each tagged polynucleotide molecule through amplification, where each copy retains the original unique identifier. This copying approach allows statistical analysis across multiple replicates while maintaining the ability to track and verify individual molecule sequences, preserving accuracy in high-throughput conditions.
3Measurement precision
If separation techniques are used for chain termination sequencing, then measurement precision is improved, but device complexity and processing time increase
Solution Approach 1:
The invention extracts the separation step from the sequencing process by using unique molecular identifiers to track individual molecules through sequencing-by-ligation or sequencing-by-synthesis without requiring physical separation of chain termination products. This extraction of the separation function maintains measurement precision through molecular tagging while dramatically reducing device complexity.
Solution Approach 2:
The invention replaces the mechanical separation system (electrophoresis, capillary separation) with an information-based system using unique molecular identifiers. Instead of physically separating molecules by size or charge, the system uses digital tracking of tagged molecules, substituting a complex mechanical separation apparatus with simpler sequencing chemistry and computational analysis.
Data Source
AI summary
Embodiments of the invention herein described relate to multiplex polynucleotide sequence analysis without the use of size separation methods or blotting. In certain particulars the invention relates to multiplex sequencing using massively parallel sequencing methods, such as pyrosequencing methods and sequencing by synthesis. The invention provides increased throughput, increased accuracy of enumerating sample components, and the ability to analyze greater numbers of samples simultaneously or serially on presently available systems, as well as others yet to be developed. In certain of its embodiments the invention relates to the analysis of complex microbial communities, particularly to in-depth analysis thereof in large numbers of samples.