Dark-Cycle Sequencing for Longer Reads and Rare Mutation Detection
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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 length of nucleic acid fragments, requiring expensive and laborious assembly processes.
Innovation Solution
The method involves alternating sequencing cycles with dark cycles, where nucleotide incorporation occurs without detection, allowing for extended polynucleotide growth, followed by detection in subsequent cycles, using labeled and unlabeled nucleotides with and without reversible terminators.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If traditional sequencing-by-synthesis methodologies are used with detection in every cycle, then nucleotide incorporation can be monitored, but the fragment length remains short (35-600 base pairs) making assembly laborious and expensive
Solution Approach 1:
The method performs preliminary extension actions during dark cycles where nucleotides are incorporated without detection. This allows the polynucleotide to grow longer before detection occurs, enabling sequencing of longer fragments without requiring laborious assembly processes
Solution Approach 2:
The sequencing method alternates between detection cycles and dark cycles in a periodic manner. During detection cycles, fluorescence is measured to identify incorporated nucleotides. During dark cycles, extension continues without detection, allowing accumulation of multiple nucleotides. This periodic alternation enables longer fragment sequencing while maintaining the ability to read the sequence
2Measurement precision
If detection is performed in every sequencing cycle, then nucleotide identity can be identified, but the process is time-consuming and limits fragment length
Solution Approach 1:
The method implements periodic detection where fluorescence measurement occurs only in detection cycles, while dark cycles perform extension without detection. This periodic pattern reduces the total time required compared to detecting every single nucleotide incorporation, while still maintaining sufficient measurement precision through the detection cycles
Solution Approach 2:
The extension reaction continues continuously through both detection cycles and dark cycles without interruption. The polymerase remains active and incorporates nucleotides during dark cycles even though detection is paused, maintaining the continuity of the useful extension action while reducing overall sequencing time
3Quantity of substance
If short nucleic acid fragments are sequenced, then sequencing cost decreases, but accuracy in mapping homopolymeric sequences and detecting SNPs deteriorates
Solution Approach 1:
The dark cycles perform preliminary extension actions that allow the polynucleotide to accumulate multiple nucleotides including homopolymeric regions before detection occurs. This preliminary extension without detection enables accurate resolution of homopolymeric sequences and SNP regions while maintaining cost-effectiveness
Solution Approach 2:
The sequencing process is segmented into detection cycles and dark cycles. The dark cycles segment the extension process to allow continuous growth without the constraints of continuous detection, enabling accurate sequencing of challenging regions like homopolymers and SNPs at lower cost
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, improving the resolution of complex regions with mutations or repetitive sequences, reducing costs and time, and enhancing the detection of SNPs and rare mutations.
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.