Multi-Priming Sequencing via Primer Segmentation
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Solution Overview
Problem
Current sequencing technologies require laborious efforts to analyze samples with high throughput and efficiency, and there is a need to selectively sequence multiple distinct regions of a template nucleic acid or exclude unnecessary regions, as long sequencing reads can have poor quality and include unwanted sequence information.
Innovation Solution
A method involving hybridizing primer molecules to specific target regions of a nucleic acid strand, where one primer is inactivated for extension, allowing for selective sequencing of distinct regions by activating and extending the other primer, using a support like a bead immobilized on a substrate with individually addressable locations, and employing blocking moieties and cleavage sites to control primer extension.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a single long sequencing read is obtained to cover multiple sequence regions, then the sequencing throughput is improved, but the sequencing quality deteriorates and unnecessary sequence information is included
Solution Approach 1:
The patent segments the sequencing process by using multiple separate primer molecules, each targeting specific regions of the template nucleic acid. Instead of obtaining one long read, the method performs multiple shorter sequencing reads from different starting positions, thereby improving quality while maintaining throughput through parallel processing of multiple regions.
Solution Approach 2:
The patent applies local quality by selectively sequencing only specific regions of interest within the template nucleic acid using region-specific primer molecules. This allows high-quality sequencing of particular segments while excluding unnecessary sequence information from other regions, optimizing both quality and efficiency.
2Quantity of substance
If a single long sequencing read is obtained to cover multiple sequence regions, then the sequencing coverage is improved, but the time required for laborious processing increases
Solution Approach 1:
The patent employs preliminary action by pre-synthesizing multiple primer molecules with specific sequences that correspond to different target regions before the sequencing process. This preparation allows the sequencing to proceed in parallel across multiple regions simultaneously, reducing processing time while maintaining comprehensive coverage.
Solution Approach 2:
By segmenting the sequencing task into multiple independent primer-based reactions targeting different regions, the patent enables parallel processing that reduces total processing time compared to a single sequential long read approach, while still achieving comprehensive coverage through the combined results of multiple reads.
3Adaptability or versatility
If multiple primer molecules are used to target different regions, then the selectivity of sequencing is improved, but the device complexity increases
Solution Approach 1:
The patent achieves universality by designing a system where multiple primer molecules can be used with a single sequencing platform. The primers are designed to be compatible with standard sequencing chemistry, allowing the same device to perform selective sequencing of different regions by simply changing the primer set, thereby maintaining adaptability without proportionally increasing device complexity.
Solution Approach 2:
The patent segments the sequencing function into modular primer-based units that can be independently designed and combined. This modular approach allows selective sequencing of different regions by choosing appropriate primers while using the same underlying sequencing machinery, thus improving selectivity without requiring a completely complex new system.
4Speed
If primer extension is allowed to proceed uninterrupted, then the sequencing speed is improved, but the ability to control and terminate extension at specific points is lost
Solution Approach 1:
The patent applies periodic action by using reversible terminators that allow the sequencing process to proceed in controlled cycles. The terminators can be removed at specific points to allow continuation, enabling precise control over when extension occurs and when it should stop, while maintaining high speed through efficient cycling of the termination and removal steps.
Solution Approach 2:
The patent utilizes parameter changes by employing reversible terminators that alter the chemical state of the primer extension process. The terminators can be chemically modified or removed to change the extension state from blocked to active, providing precise control over the sequencing process at specific points while maintaining overall speed through rapid reversible transitions.
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
Enables efficient and selective sequencing of specific regions of a nucleic acid, reducing unnecessary sequencing and improving sequencing quality by allowing for precise control over primer extension and data generation.
Implementation Method 1
hybridizing a first primer molecule and a second primer molecule to a first target region and a second target region, respectively, of a target nucleic acid strand
Implementation Method 2
extending the first primer molecule to generate a first sequencing read for a first region of the target nucleic acid strand
Data Source
AI summary
Provided herein are systems, methods, compositions, and kits for sequencing with multi-priming. In some cases, multiple distinct primer molecules may be provided to a template nucleic acid to hybridize to distinct regions of the template nucleic acid. In some cases, a connected primer molecule may be provided to a template nucleic acid to have multiple distinct primer regions hybridize to distinct regions of the template nucleic acid. Non-adjacent regions of the template nucleic acid may be sequenced in distinct sequencing operations.