Long Nucleic Acid Sequencing via Primer Extension Segmentation
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Solution Overview
Problem
Current nucleic acid sequencing methods face limitations in efficiency and length due to stepwise inefficiencies such as incomplete incorporation and ligation, leading to prephasing or dephasing, which restrict read length and quality.
Innovation Solution
The method involves controlled extension of sequencing primers using reversible terminator nucleotides and pulse extension techniques, allowing for longer sequence reads by assembling multiple sequence segments obtained from different primers, which can be performed on a substrate like a flow cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If stepwise sequencing methods (polymerase extension or ligation) are used to sequence nucleic acids, then sequencing can be performed with existing technology, but read length is limited due to stepwise inefficiencies such as incomplete incorporation and ligation causing prephasing or dephasing
Solution Approach 1:
The patent divides the sequencing process into distinct segments: (1) primer extension phase where primers are extended along the template, and (2) sequencing phase where the extended primers are read. By separating these functions and allowing primer extension to proceed without the constraints of stepwise incorporation, the method achieves longer read lengths while maintaining accuracy through the dedicated sequencing read step.
Solution Approach 2:
The patent performs preliminary primer extension before sequencing, allowing the primers to fully extend along the template strand in advance. This preliminary action creates complete complementary strands that can then be read sequentially, enabling longer reads by eliminating the stepwise limitation during the actual sequencing phase.
2Length of moving object
If controlled extension with reversible terminator nucleotides is used, then longer contiguous sequences can be achieved, but the process complexity increases
Solution Approach 1:
The patent extracts the extension function from the sequencing read function. By using primer extension to create complementary strands and then separately reading these extended primers, the method removes the stepwise incorporation constraint that traditionally linked extension and sequencing, thereby enabling longer reads without proportionally increasing overall process complexity.
3Quantity of substance
If multiple sequence segments from different primers are assembled to extend read length, then sequencing coverage is improved, but the assembly process requires additional computational and experimental steps
Solution Approach 1:
The patent employs universal primer binding sites that allow multiple primers to bind to different locations on the template strand. These universal sites enable systematic coverage of the entire template through multiple priming events, with the extended primers being assembled into a complete sequence, thereby achieving comprehensive coverage through a standardized multi-functional approach.
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 extends the read length beyond traditional sequencing limitations, achieving longer contiguous sequences with improved quality scores, enabling more accurate and efficient sequencing of nucleic acids.
Implementation Method 1
a first sequencing primer is hybridized to a target nucleic acid
Implementation Method 2
extending a first sequencing primer hybridized to the target nucleic acid to generate a first primer extension product
Implementation Method 3
The target nucleic acid and the primer extension product can be denatured and the primer extension product released
Data Source
AI summary
The present invention provides methods and systems for sequencing long nucleic acid fragments. In one aspect of the invention, methods, systems and reagent kits are provided for sequencing nucleic acid target sequences. Some embodiments of the methods, systems and reagent kits are particularly suitable for sequencing a large number of fragments, particularly long fragments.