Nucleic Acid Capping Chemistry for Low-Phasing Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing techniques using ensembles suffer from phasing issues that limit read length, throughput, and accuracy due to out-of-sync primer molecules and increased background noise, leading to reduced sequencing quality.
Innovation Solution
The method involves forming ternary complexes by incorporating blocked or reversibly terminated nucleotides and ternary complex inhibitors at the 3′ end of primers, allowing for selective detection of nucleotides without forming unwanted background signals, thereby preventing phasing and improving sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If ensemble-based sequencing techniques are used to process multiple nucleic acids in parallel, then sequencing throughput and productivity are improved, but phasing occurs causing loss of signal accuracy and read length limitations
Solution Approach 1:
The patent extracts and removes the harmful phasing effect by introducing a mechanism to eliminate out-of-sync primer molecules from the ensemble. Specifically, it uses a combination of selective primer extension and removal techniques to take out only the problematic primers that cause phasing, while retaining the synchronized primers that provide accurate signals.
Solution Approach 2:
The patent applies local quality by differentiating the fate of different primers within the ensemble based on their synchronization status. Synchronized primers are retained for accurate sequencing, while out-of-sync primers are selectively removed. This creates different qualities of primer treatment within the same sequencing process.
2Loss of time
If multiplex sequencing is used to process large collections of nucleic acids, then time and reagent costs are reduced, but side reactions and incomplete reactions create artifacts and background signals
Solution Approach 1:
The patent converts the harmful side reactions and incomplete reactions that occur during multiplex sequencing into beneficial selective removal mechanisms. By designing the sequencing chemistry to exploit these side reactions, it selectively removes unwanted primers and artifacts while maintaining desired sequencing reactions, thereby converting harm into benefit.
Solution Approach 2:
The patent introduces intermediary substances or steps that mediate between the multiplex sequencing process and the final detection. These intermediaries include selective extension reagents and removal agents that facilitate the differentiation and removal of problematic species without interfering with the overall multiplex process.
3Length of moving object
If phasing occurs at a rate of 0.5% per cycle, then read length is limited due to accumulated signal loss, but increasing read length would exacerbate phasing accumulation
Solution Approach 1:
The patent applies preliminary action by performing selective primer removal and synchronization steps before the main sequencing cycles begin. This preliminary processing eliminates phasing-prone primers in advance, preventing the accumulation of phasing errors during subsequent read cycles and enabling longer reads with maintained signal-to-noise ratios.
Solution Approach 2:
The patent implements feedback mechanisms that continuously monitor and adjust the sequencing process to compensate for phasing. By detecting phasing events and applying corrective measures in real-time, the system maintains signal accuracy even as read length increases, breaking the trade-off between read length and signal quality.
Data Source
AI summary
A method for identifying a nucleic acid template that include (a) providing a plurality of primer-template hybrids, wherein a first hybrid of the plurality includes a first template hybridized to a first primer, and wherein a second hybrid of the plurality includes a second template hybridized to a second primer, the second primer having a ternary complex inhibitor moiety at the 3′ end; (b) delivering polymerases and nucleotides to the plurality, whereby the first hybrid binds a polymerase and nucleotide to form a stabilized ternary complex and whereby the second hybrid does not bind a polymerase and nucleotide to form a stabilized ternary complex; and (c) detecting the stabilized ternary complex to identify the first template.