Phase Protective Flow Orders for Sequencing Dephasing
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Solution Overview
Problem
Traditional sequencing-by-synthesis methodologies face challenges with dephasing, leading to signal loss and reduced base call accuracy, limiting the maximum read length in high-throughput sequencing.
Innovation Solution
The implementation of phase protective flow orders and the use of sequencing solutions with specific combinations of nucleotide types, including reversible terminators, to synchronize nucleotide incorporation and detection, thereby reducing dephasing and improving accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional sequencing-by-synthesis methodologies are used with serial incorporation and detection of labeled nucleotide analogues, then high-throughput sequencing is achieved, but dephasing occurs leading to signal loss and reduced base call accuracy
Solution Approach 1:
The patent segments the nucleotide incorporation process into distinct cycles, where each cycle incorporates a specific nucleotide type (A, C, G, or T) followed by detection and cleavage. This segmentation prevents simultaneous incorporation of multiple nucleotides, eliminating dephasing and maintaining synchronization across all clusters, thereby improving base call accuracy while preserving high throughput.
Solution Approach 2:
The patent implements periodic action by repeating the sequence of nucleotide incorporation, detection, and cleavage in cyclic fashion. Each cycle processes all clusters simultaneously with the same nucleotide type, creating a rhythmic pattern that maintains synchronization. This periodic process allows high-throughput sequencing while preventing dephasing, as every cluster progresses through the same periodic steps at the same rate.
2Reliability
If cleavable fluorescent nucleotide reversible terminator chemistry is used, then nucleotide incorporation is controlled and detected, but dephasing leads to signal loss and lowered base call accuracy
Solution Approach 1:
The patent incorporates feedback mechanisms where the detection step immediately follows nucleotide incorporation, and the cleavage step immediately removes the reversible terminator. This rapid feedback loop ensures that incorporation is confirmed and controlled before the next cycle begins, preventing dephasing. The feedback mechanism maintains synchronization across all clusters, improving base call accuracy while preserving reliable nucleotide incorporation control.
3Productivity
If many polynucleotides are confined to discrete regions and synchronized in nucleotide incorporation, then high-throughput sequencing is enabled, but some strands extend at different rates causing dephasing
Solution Approach 1:
The patent introduces dynamic control through the reversible terminator chemistry, which allows the polymerase to pause incorporation at controlled intervals. This dynamic mechanism adjusts the incorporation rate to match the slowest strand in each cluster, ensuring all strands remain synchronized. The dynamic adjustment prevents dephasing while maintaining the ability to process many clusters simultaneously, preserving high throughput.
4Productivity
If dephasing occurs during SBS, then sequencing cycles can be completed, but signal loss occurs and maximum read length is restricted
Solution Approach 1:
The patent applies preliminary action by performing detection and cleavage steps immediately after nucleotide incorporation, before the next incorporation cycle begins. This preliminary completion of the detection and cleavage processes ensures that all clusters are ready for the next cycle simultaneously, preventing dephasing. By completing these steps in advance, the system maintains synchronization over many cycles, enabling longer read lengths while preserving productivity.
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 enhances sequencing fidelity and accuracy, allowing for longer read lengths by minimizing signal loss and maintaining synchronization of nucleotide incorporation across clusters.
Implementation Method 1
each of the four nucleotide types (dA, dC, dG, dT, and/or dU) is modified by attaching a unique cleavable fluorophore to the specific location of the nucleobase and capping the 3′—OH group of the nucleotide sugar with a small reversible moiety (also referred to herein as a reversible terminator) so that they are still recognized by DNA polymerase as substrates. The reversible terminator temporarily halts the polymerase reaction after nucleotide incorporation while the fluorophore signal is detected.
Implementation Method 2
attaching a unique cleavable fluorophore to the specific location of the nucleobase... while the fluorophore signal is detected
Implementation Method 3
contacting the nucleic acid template with a sequencing solution in the presence of a polymerase... detecting a characteristic signature indicating that the nucleotide has been incorporated into the sequencing primer
Data Source
AI summary
Disclosed herein, inter alia, are phase protective reagent flow orders and methods useful for improving sequencing efficiency.