Time-Resolved Nucleic Acid Sequencing via Intermediate Signal Detection
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Solution Overview
Problem
Current nucleic acid sequencing methods based on end-point measurements are limited by long incubation and reaction times, leading to speed and throughput issues, and are prone to false-read errors due to misinterpretation of auto-fluorescence and unspecific incorporation of fluorescently labeled oligonucleotides.
Innovation Solution
A nucleic acid sequencing method that involves contacting a target nucleic acid sequence with multiple oligonucleotide probes and performing time-resolved measurements during hybridization or ligation reactions, co-processing data sets to identify labels or their absence, and determining the sequenced base based on these measurements, thereby eliminating the need for end-point measurements and reducing false-read errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If end-point measurements are used in sequencing reactions, then measurement simplicity is maintained, but sequencing speed and throughput are severely limited due to long incubation times (30-60 min) required to achieve sufficient signal-to-noise ratio
Solution Approach 1:
The patent applies preliminary action by performing multiple measurements during the hybridization/ligation reaction process before the reaction reaches completion. Instead of waiting for the full 30-60 minute incubation period to achieve sufficient signal accumulation, the system takes measurements at intermediate time points (e.g., at 10, 20, 30 minutes), allowing early detection of correctly incorporated oligonucleotides and significantly reducing the total sequencing time while maintaining adequate measurement precision
Solution Approach 2:
The patent implements feedback by using the results from intermediate measurements to guide subsequent sequencing steps. The measurement results from earlier time points provide feedback about which oligonucleotides have been correctly incorporated, allowing the system to adjust reaction conditions, extend incubation time selectively for ambiguous cases, or proceed to the next sequencing cycle, thereby optimizing both speed and precision dynamically
2Measurement precision
If end-point measurements are performed after long incubation periods, then sufficient signal accumulation is achieved, but false-read errors increase due to misinterpretation of auto-fluorescence and unspecific incorporation
Solution Approach 1:
The patent applies preliminary action by performing measurements at multiple intermediate time points during the hybridization/ligation reaction rather than waiting for the reaction to complete. This allows the system to detect correctly incorporated oligonucleotides before unspecific binding and auto-fluorescence accumulate to problematic levels, thereby maintaining high measurement precision while improving sequencing reliability by avoiding false-read errors
Solution Approach 2:
The patent applies the skipping principle by rushing through the measurement process at optimized intermediate time points rather than completing the full incubation period. By taking measurements at strategically chosen intermediate moments when specific incorporation is maximized but unspecific background is still low, the system skips the problematic later stages where false-read errors would occur, achieving both accurate detection and high reliability
3Measurement precision
If removal and washing steps are included in the sequencing protocol, then background noise is reduced and signal-to-noise ratio is improved, but processing time and operational complexity increase significantly
Solution Approach 1:
The patent applies the taking out principle by extracting and eliminating the removal and washing steps from the traditional sequencing protocol. By performing measurements during the hybridization/ligation reaction itself, the system removes the need for separate washing steps that would otherwise be required to remove unincorporated oligonucleotides. This extraction of unnecessary steps significantly reduces processing time while maintaining adequate signal-to-noise ratio through the use of appropriate controls and measurement timing
Solution Approach 2:
The patent applies continuity of useful action by maintaining the hybridization/ligation reaction conditions throughout the measurement process without interrupting the reaction for removal or washing steps. The measurement process continues uninterrupted alongside the chemical reaction, allowing the useful action of oligonucleotide incorporation to proceed continuously while measurements are taken at intermediate time points, thereby eliminating time loss to auxiliary steps
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 speeds up the sequencing process and improves accuracy by performing measurements during the reaction, reducing the risk of false-read errors and eliminating the need for lengthy incubation and washing steps.
Implementation Method 1
a hybridization reaction comprising hybridization of the N oligonucleotide probes to the target nucleic acid sequence
Implementation Method 2
a ligation reaction comprising ligation of the N oligonucleotide probes to an anchor probe
Implementation Method 3
The method also comprises performing measurements, at least at the spatially defined site, at M≥2 time instances during i) a hybridization reaction comprising hybridization of the N oligonucleotide probes to the target nucleic acid sequence
Data Source
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AI summary
A nucleic acid sequencing method involving contacting a spatially defined target nucleic acid sequence with N labeled oligonucleotide probes and performing measurements, at least at the spatially defined site (1), at M time instances during ligation or hybridization to form M data sets. The M data sets are coprocessed in order to identify a label or absence of any label at the spatially defined site (1). A sequenced based of the target nucleic acid sequence is then determined based on the identified label of the identified absence of any label. The time-resolved measurements conducted during the actual hybridization of the oligonucleotide probes to the target nucleic acid sequence or the ligation of oligonucleotide probes to anchor probes on the target nucleic acid sequence improves the speed and accuracy of the sequencing as compared to the state of the art sequencing methods.