Nucleic Acid Synchronization via Incomplete Nucleotide Sets
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Solution Overview
Problem
Current nucleic acid sequencing methods face limitations in achieving long, accurate, and cost-effective sequencing, particularly for point-of-care applications and field detection of pathogens, due to inefficiencies in stepwise sequencing methods that lead to prephasing or dephasing, resulting in reduced read length and quality.
Innovation Solution
The implementation of a sequencing-by-synthesis system with synchronization steps using incomplete sets of nucleotides, such as dATP, dCTP, dGTP, and dTTP, to resynchronize sequencing products, thereby improving chastity scores and extending accurate base calls, allowing for longer sequencing reads with higher accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If stepwise sequencing methods are used, then sequencing can be performed, but prephasing or dephasing occurs resulting in reduced read length and quality
Solution Approach 1:
The patent implements periodic synchronization steps interspersed among sequencing steps. The synchronization steps use incomplete nucleotide sets (missing one or more dNTPs) to periodically reset and realign the extension states of nascent strands, counteracting the cumulative dephasing that occurs during continuous stepwise sequencing. This periodic intervention restores synchronization without completely halting the sequencing process.
Solution Approach 2:
The patent changes the nucleotide composition parameters during the sequencing process by introducing synchronization steps with incomplete nucleotide sets. By temporarily removing specific dNTPs from the reaction mixture during synchronization steps, the system alters the extension kinetics and forces strands into a synchronized state, thereby improving both read length and quality metrics.
2Productivity
If stepwise sequencing methods are used, then sequencing can be performed, but prephasing or dephasing occurs resulting in reduced quality
Solution Approach 1:
The patent implements periodic synchronization steps interspersed among sequencing steps. The synchronization steps use incomplete nucleotide sets (missing one or more dNTPs) to periodically reset and realign the extension states of nascent strands, counteracting the cumulative dephasing that occurs during continuous stepwise sequencing. This periodic intervention restores synchronization without completely halting the sequencing process.
Solution Approach 2:
The patent maintains continuous sequencing action by performing multiple sequencing steps between synchronization events. Rather than stopping sequencing entirely for synchronization, the method continues productive sequencing at 75-90% capacity between sync steps, then periodically intervenes to restore quality, thereby maintaining both throughput and quality over extended read lengths.
3Manufacturing precision
If synchronization steps with incomplete nucleotide sets are used, then chastity scores improve, but additional steps are required
Solution Approach 1:
The patent implements periodic synchronization steps interspersed among sequencing steps. The synchronization steps use incomplete nucleotide sets (missing one or more dNTPs) to periodically reset and realign the extension states of nascent strands, counteracting the cumulative dephasing that occurs during continuous stepwise sequencing. This periodic intervention restores synchronization without completely halting the sequencing process.
Solution Approach 2:
The synchronization steps serve multiple functions: they synchronize nascent strands, improve chastity scores, and enable extended read lengths without requiring separate dedicated synchronization procedures. The same incomplete nucleotide set mechanism achieves both quality improvement and length extension goals simultaneously.
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 significantly enhances the length and accuracy of sequencing reads, achieving chastity scores of at least 0.85 or greater and increasing the length of accurate base calls by up to 500 base pairs, while reducing errors and improving synchronization of sequencing strands.
Implementation Method 1
extending the plurality of sequencing products with a DNA polymerase in the presence of a set of up to three different nucleotides
Implementation Method 2
hybridization to a target and ligated together
Data Source
AI summary
The methods, systems, kits and reagents provided herein relate to the synchronization of dephased/prephased nucleic acid molecules during sequencing reactions. The methods, systems, kits and reagents provided herein can be utilized to improve the efficiency and accuracy of sequencing technologies.