Modified Polynucleotide Preparation for Clearer Nanopore Sequencing
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Solution Overview
Problem
Existing polynucleotide sequencing technologies are slow and expensive due to reliance on amplification techniques and require high quantities of specialist fluorescent chemicals, and nanopore sequencing faces challenges in resolving current measurements for different k-mers, especially at high values of k, leading to difficulty in deriving accurate sequence information.
Innovation Solution
A modified polynucleotide is created by replacing one or more nucleotide species in the template polynucleotide with different nucleotide species using a polymerase, which is then characterized using a transmembrane pore, providing distinct current measurements that enhance sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If nanopore sequencing is used to directly detect polynucleotide sequences, then the need for amplification techniques and fluorescent chemicals is reduced, but the ability to resolve current measurements for different k-mers deteriorates, especially at high values of k
Solution Approach 1:
The patent introduces a modified polynucleotide as an intermediary between the template polynucleotide and the nanopore detection system. This modified version contains altered k-mers that produce distinct current measurements, serving as a mediator that bridges the gap between the original sequence and the detection capability of the nanopore system.
Solution Approach 2:
The patent applies parameter changes by modifying the chemical composition of nucleotides within k-mers to create altered k-mers with different electrical properties. These parameter changes in nucleotide composition result in distinct current blockage patterns that improve measurement resolution without requiring additional fluorescent chemicals.
2Loss of information
If k-mer size is increased to improve sequencing coverage, then more information is captured per measurement, but the separation between current measurements for different k-mers decreases, making it difficult to resolve measurements
Solution Approach 1:
The patent modifies the chemical parameters of nucleotides within k-mers to create altered k-mers with distinct electrical signatures. This allows larger k-mer sizes to be used for comprehensive information capture while maintaining measurement separation through the modified nucleotide compositions that produce unique current blockage patterns.
Solution Approach 2:
The patent segments the polynucleotide sequence into modified k-mers with distinct compositional characteristics. By creating altered k-mers with specific nucleotide compositions, each segment produces a unique current measurement signature, enabling better resolution even when larger k-mer sizes are used for increased information capture.
3Quantity of substance
If amplification techniques are used to produce large volumes of polynucleotide, then sufficient material for sequencing is obtained, but the process becomes slow and expensive
Solution Approach 1:
The patent employs polymerases that can directly synthesize modified polynucleotides from template polynucleotides without requiring complex amplification techniques. The polymerase system self-services by incorporating modified nucleotides during synthesis, producing sufficient material for sequencing while maintaining rapid processing speeds and reducing costs.
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
The modified polynucleotide offers clearer and more separable current measurements, facilitating easier characterization of the template polynucleotide, improving sequencing accuracy and reducing the need for costly fluorescent chemicals.
Implementation Method 1
The polymerase uses the template polynucleotide as a template to form a modified polynucleotide from the population of free nucleotides
Implementation Method 2
When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time
Data Source
AI summary
The invention relates to an improved method for characterising a template polynucleotide. The method involves using a polymerase to prepare a modified polynucleotide which makes it easier to characterise that the template polynucleotide.