De Bruijn Reference Maps for Nanopore Signal Resolution
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Solution Overview
Problem
Existing nanopore-based methods struggle to achieve single-nucleotide resolution in polymer sequencing due to multi-subunit output signals, leading to difficulties in correlating observed signals with polymer characteristics and inconsistencies across assays.
Innovation Solution
A method involving the translocation of a reference sequence through a nanopore to generate a reference map, utilizing a De Bruijn sequence optimized for multi-subunit output signals, allowing each possible multi-subunit sequence to appear only once, and compiling these signals into a reference map for analyte polymer analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nanopore-based methods are used to analyze polymers, then the analysis can be performed with existing nanopore systems, but the output signals represent multiple subunits simultaneously making single-nucleotide resolution difficult to achieve
Solution Approach 1:
The patent segments the polymer analysis problem by using a De Bruijn sequence that systematically divides all possible multi-subunit combinations into unique, non-redundant segments. Each segment of the reference sequence corresponds to a specific multi-subunit combination, allowing the complex multi-subunit signal problem to be broken down into manageable, uniquely-mapped segments that can be individually characterized and assembled into a complete reference map.
Solution Approach 2:
The patent introduces a De Bruijn sequence as an intermediary reference structure that mediates between the multi-subunit nanopore signals and the desired single-nucleotide resolution. This intermediary sequence provides a systematic framework that maps each possible multi-subunit combination to a unique position, acting as a bridge that translates the complex multi-subunit output into resolvable single-nucleotide information.
2Adaptability or versatility
If a comprehensive reference sequence containing all possible multi-subunit sequences is generated, then complete coverage of all polymer variations is achieved, but the length of the reference sequence becomes excessively long increasing synthesis costs and efforts
Solution Approach 1:
The De Bruijn sequence segments the complete set of all possible multi-subunit sequences into a compact, non-redundant structure. Instead of listing every possible combination separately (which would be exponentially long), the sequence segments overlaps efficiently so that each unique multi-subunit combination appears exactly once, minimizing the total length while maintaining complete coverage.
Solution Approach 2:
The patent transforms the problem from a linear enumeration of all possible sequences into a dimensional structure where the De Bruijn sequence of order n on k symbols provides complete coverage in a compact space. This dimensional approach allows the reference sequence to encode all possible multi-subunit combinations of length n using only k^n + n - 1 symbols, rather than requiring separate representations for each combination.
3Loss of information
If deconvolution steps are used to deduce correlation between observed signals and physical characteristics of monomeric subunits, then sequence information can be extracted, but the process becomes complex and susceptible to errors from signal fluctuations
Solution Approach 1:
The patent performs preliminary action by pre-generating a De Bruijn reference sequence that contains all possible multi-subunit combinations in a systematic, non-redundant manner. This reference sequence is processed beforehand to create a lookup table or reference map that directly correlates each possible signal pattern with its corresponding sequence information, eliminating the need for complex real-time deconvolution and reducing susceptibility to signal fluctuations.
Solution Approach 2:
The patent creates a complete reference copy of all possible multi-subunit sequences using the De Bruijn structure. This reference copy serves as a template against which experimental signals can be directly compared, replacing the need for complex mathematical deconvolution with a simpler pattern-matching approach that is more robust to signal variations.
4Adaptability or versatility
If minor fluctuations in assay conditions or nanopore characteristics occur, then natural variations in the system are introduced, but these fluctuations differentially influence monitored signals making comparisons between assays difficult
Solution Approach 1:
The patent uses parameter changes by systematically varying the composition of multi-subunit sequences in the De Bruijn reference sequence to cover all possible combinations. This comprehensive parameter sampling creates a reference map that accounts for natural variations in assay conditions and nanopore characteristics, allowing experimental data to be normalized against this complete reference set and improving assay consistency across different runs and systems.
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 enhances the ability to accurately determine polymer sequences by minimizing the length of the reference sequence, reducing synthesis costs and efforts, and improving assay consistency across different nanopore systems.
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.