Dark-Cycle Sequencing for Longer Reads in Complex DNA Regions
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Solution Overview
Problem
Traditional sequencing technologies face challenges in accurately mapping homopolymeric sequences, detecting single nucleotide polymorphisms (SNPs), and identifying rare mutations due to the relatively short lengths of nucleic acid fragments, requiring expensive and laborious Sanger sequencing methods and de novo assembly of complex regions with mutations or repetitive sequences.
Innovation Solution
The method involves alternating sequencing cycles with dark cycles, where nucleotide incorporation occurs without detection, allowing for extended polynucleotide extension, followed by detection in subsequent cycles, using labeled and unlabeled nucleotides with reversible terminators to enhance sequencing accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing-by-synthesis methodologies are used with detection in every cycle, then nucleotide incorporation can be identified, but sequencing time and cost increase significantly for long templates
Solution Approach 1:
The patent implements periodic detection cycles interspersed with dark cycles (no detection). Detection occurs at specific intervals rather than continuously, reducing the temporal overhead of detection operations while still capturing sufficient information to resolve the sequence. This periodic action allows the system to balance detection accuracy with reduced sequencing time.
Solution Approach 2:
The patent performs preliminary nucleotide incorporation during dark cycles before detection cycles. By extending polynucleotides in advance during dark cycles and then detecting in subsequent cycles, the system prepares the molecular state beforehand, allowing faster overall sequencing while maintaining detection accuracy when it occurs.
2Productivity
If de novo assembly of short DNA fragments is performed, then sequencing can be achieved, but resolving complex regions with mutations or repetitive sequences becomes difficult
Solution Approach 1:
The patent segments the sequencing process into alternating detection cycles and dark cycles, where dark cycles enable extended polynucleotide extension. This segmentation allows the system to accumulate longer extension products that span complex regions, improving resolution of repetitive sequences and mutations while maintaining high throughput through parallel processing.
Solution Approach 2:
The patent dynamically adjusts the sequencing approach by alternating between detection and non-detection cycles. This dynamic protocol allows the system to adaptively extend polynucleotides through complex regions during dark cycles and then verify incorporations during detection cycles, improving accuracy in difficult-to-sequence regions without sacrificing overall productivity.
3Measurement precision
If continuous detection of nucleotide incorporation is performed, then sequence accuracy can be maintained, but the complexity and cost of the sequencing system increase
Solution Approach 1:
The patent reduces device complexity by implementing periodic detection instead of continuous detection. The system alternates between detection cycles (with imaging) and dark cycles (without imaging), reducing the operational burden on detection instruments while maintaining sequence accuracy through strategic sampling of incorporation events.
Solution Approach 2:
The patent enables the sequencing system to self-regulate by using dark cycles for extension and detection cycles for verification. The system inherently manages its own complexity by alternating between states, reducing the need for continuous active monitoring and control mechanisms while preserving measurement precision through periodic verification.
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 enables accurate sequencing of longer template nucleic acids with improved resolution of complex regions, reducing sequencing time and cost while enhancing the detection of mutations and repetitive sequences.
Implementation Method 1
extending a complementary polynucleotide that is hybridized to the template nucleic acid by incorporating a first nucleotide using a polymerase
Implementation Method 2
detecting a label that identifies the first nucleotide
Data Source
AI summary
Provided herein are methods including alternating series of sequencing cycles and dark extension cycles allowing longer read lengths and addressing disadvantages of traditional nucleic acid sequencing protocols.