Nanopore Reference Maps Using De Bruijn Sequences for Polymer Signals
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Solution Overview
Problem
Existing nanopore-based methods struggle to achieve single-nucleotide resolution in polymer sequencing due to multi-subunit interactions within the nanopore constriction zone, leading to complex signal correlations and assay variability.
Innovation Solution
A method is developed to generate a reference map using a reference sequence that includes every possible multi-subunit polymer segment only once, allowing for efficient correlation of output signals with polymer sequences, using De Bruijn sequences and optimized nanopore systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nanopore-based methods are used for polymer sequencing, then the analysis can be performed with existing nanopore systems, but single-nucleotide resolution cannot be achieved due to multi-subunit interactions within the nanopore constriction zone
Solution Approach 1:
The patent segments the polymer analysis problem by focusing on multi-subunit sequences rather than individual nucleotides. The reference map is constructed using multi-subunit sequences (e.g., 4-mer, 5-mer, 6-mer) that pass through the nanopore constriction zone together, allowing the system to resolve sequences at multi-nucleotide resolution despite the physical constraints of the nanopore geometry.
Solution Approach 2:
The patent introduces a reference map as an intermediary data structure that correlates multi-subunit sequences with their corresponding electrical signals. This reference map serves as a lookup table that simplifies the complex signal correlation process by pre-establishing the relationship between known multi-subunit sequences and their signal characteristics, enabling accurate polymer sequencing without requiring real-time deconvolution of complex signals.
2Reliability
If reference sequences are generated using traditional methods, then comprehensive coverage of polymer sequences can be achieved, but the length and cost of reference sequence synthesis increase substantially
Solution Approach 1:
The patent merges overlapping multi-subunit sequences into a compact reference sequence. By using algorithms that identify and combine sequences that share common multi-subunit segments, the reference sequence achieves comprehensive coverage of all possible polymer sequences while minimizing redundancy. This merging approach significantly reduces the total length of the reference sequence compared to traditional methods that would require separate sequences for each possible combination.
Solution Approach 2:
The patent creates a universal reference sequence that can serve multiple functions: it provides comprehensive sequence coverage, enables correlation of electrical signals with polymer sequences, and can be used across different nanopore-based analysis systems. The reference sequence is designed to be multi-functional, serving as both a calibration standard and a sequencing reference, thereby reducing the need for multiple separate reference sequences.
3Productivity
If nanopore-based analysis is used for polymer sequencing, then the method can provide rapid and cost-effective analysis, but assay variability and signal fluctuations make comparisons between assays difficult
Solution Approach 1:
The patent addresses assay variability by normalizing electrical signal parameters across different assays. The system adjusts for fluctuations in assay conditions by comparing signals relative to a reference map that accounts for expected signal characteristics. This parameter normalization enables reliable comparison of sequencing results between different assays, nanopores, and experimental conditions while maintaining the rapid throughput of nanopore-based analysis.
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 reduces the length and cost of reference sequence synthesis, enhances signal clarity, and enables accurate polymer sequencing by correlating multi-subunit output signals with specific polymer segments, improving sequencing efficiency and reliability.
Implementation Method 1
passing a polymeric molecule, for example single-stranded DNA (''ssDNA''), through a nanoscopic opening while monitoring a signal such as an electrical signal that is influenced by the physical properties of the target molecule as it passes through the nanopore opening
Data Source
AI summary
The present disclosure generally relates to the methods and compositions to efficiently analyze polymer characteristics using nanopore-based assays. Specifically disclosed is a method for generating reference signals for polymer analysis in a nanopore system, wherein the nanopore system has a multi-subunit output signal resolution. The method comprises translocating a reference sequence through a nanopore to generate a plurality of reference output signals, wherein each possible multi-subunit sequence that can determine an output signal appears only once in the reference sequence. The output signals are compiled into a reference map for nanopore analysis of an analyte polymer. Also provided are methods and compositions for calibrating the nanopore system for optimized polymer analysis.