Massively Parallel Sequencing Rephasing Method
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Solution Overview
Problem
In massively parallel sequencing methods, the accumulation of out-of-phase templates due to non-incorporation or misincorporation during nucleotide incorporation cycles leads to increased signal-to-noise ratio and limited read length, compromising accuracy and sequence quality.
Innovation Solution
The method involves rephasing extended primers in a clonal population of nucleic acid duplexes by extending them with nucleotide triphosphates A, T, C, and G, using a reversible terminator blocked with a first blocking group, followed by unblocking and subsequent treatment with a second mixture containing a single nucleotide triphosphate blocked with a second blocking group, until all primers are aligned, thereby reducing discordance and improving phase concordance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If multiple cycles of sequencing reaction are performed to extend read length, then more sequence information is obtained, but out-of-phase templates accumulate leading to increased error rates and reduced accuracy
Solution Approach 1:
The patent applies periodic rephasing actions during the sequencing process. After a certain number of sequencing cycles, a rephasing step is periodically introduced where all extended primers are extended by a fixed number of nucleotides (e.g., 5-10 nt) using unblocked nucleotides, then blocked with reversible terminators. This periodic rephasing resets the phase of out-of-phase templates, allowing the sequencing to continue with high accuracy for extended read lengths.
2Length of stationary object
If sequencing cycles are extended to improve read length, then more template positions are covered, but signal-to-noise ratio deteriorates due to out-of-phase accumulation
Solution Approach 1:
The periodic rephasing intervention removes out-of-phase templates from the population at regular intervals during sequencing. By extending all primers to a standardized position and blocking them, the method eliminates the accumulation of out-of-phase signals that would otherwise degrade the signal-to-noise ratio, enabling maintenance of high signal quality over extended read lengths.
Solution Approach 2:
The rephasing process effectively discards the out-of-phase information by extending primers past the point of divergence and then blocking them. The useful in-phase information is recovered by continuing sequencing from the rephased position, thereby eliminating harmful out-of-phase signals while preserving and extending the useful sequence information.
3Reliability
If rephasing is performed using blocked nucleotides only, then phase concordance is restored, but sequencing continuity is interrupted and time is lost
Solution Approach 1:
The patent performs preliminary extension with unblocked nucleotides before blocking with reversible terminators. This preliminary action extends all primers to the same position (including those that were out-of-phase), ensuring phase concordance is achieved before the blocking step, thereby minimizing the need for additional unblocking and re-extension cycles.
Solution Approach 2:
The rephasing process maintains continuity of useful sequencing action by using unblocked nucleotides for the extension portion, allowing all primers to be extended simultaneously without interruption. The blocking step then uniformly terminates this continuous extension, preserving the continuity of the sequencing workflow while achieving phase alignment.
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 reduces discordance among sequencing primers, enhancing accuracy and extending read length by ensuring that all primers are in phase, allowing for longer sequencing reads with improved signal intensity and reduced errors.
Implementation Method 1
extending the extended primers by incorporating one or more nucleotides that are complementary to the template sequence using a polymerase and nucleotides comprising nucleotide triphosphates A, T, C, and G
Implementation Method 2
one of the nucleotides is a reversible terminator blocked with a first blocking group
Data Source
Figure 1~2
Figure 3
Figure 4A~4B
AI summary
Determining the sequence of a nucleic acid typically entails performing multiple cycles of a reaction that generates a signal, depending on the identity of one or more nucleotides in the sequence. Sequencing typically is done on a plurality of copies of a template to fortify the signal and to increase accuracy. However, as the number of cycles increases, some of the copies go out of phase, increasing signal-to-noise ratio and compromising accuracy. Provided is a strategy using blocking groups and dinucleotide recognition to bring each of the copies back into phase. This improves accuracy and enables the user to increase the length of sequence reads.