Concatemer Sequencing With Reduced Labels for Multi-Base Detection
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Solution Overview
Problem
Conventional sequencing methods are limited by signal degradation and signal-to-noise ratios, restricting sequencing efficiency and suitability for single-molecule sequencing, and often require multiple probes to distinguish between nucleotides, increasing complexity and cost.
Innovation Solution
The use of concatemers with adaptors and sequencing probes labeled with fewer than four unique labels allows for efficient base identification through hybridization and ligation, enabling sequencing-by-synthesis, sequencing-by-ligation, or cPAL methods to determine nucleotide identities with reduced probe sets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional sequencing methods are used, then sequencing can be performed, but signal degradation limits the number of nucleotides that can be determined and reduces sequencing efficiency
Solution Approach 1:
The target sequence is divided into multiple concatemers, each containing multiple monomers with target domains. This segmentation allows parallel sequencing of multiple segments simultaneously, increasing overall productivity while maintaining signal quality through repeated sequencing of the same target from different concatemer instances
Solution Approach 2:
Multiple copies of the target sequence are created through concatemer formation, where each concatemer contains multiple monomers with identical target domains. These copies enable parallel analysis and improve sequencing efficiency without degrading signal quality, as each copy can be independently sequenced
2Productivity
If conventional sequencing methods are used, then sequencing can be performed, but poor signal-to-noise ratios make single-molecule sequencing unsuitable
Solution Approach 1:
Multiple signal sources are merged by sequencing the same target sequence from multiple concatemer copies simultaneously. This combining of signals from parallel reactions improves the effective signal-to-noise ratio, enabling reliable single-molecule sequencing that would otherwise be unsuitable due to poor signal quality
Solution Approach 2:
Multiple copies of the target are sequenced in parallel, and the resulting signals are combined. This copying approach amplifies the effective signal while averaging out noise, improving measurement precision and enabling single-molecule sequencing capability
3Measurement precision
If multiple probes are used to distinguish between nucleotides, then nucleotide identification can be achieved, but probe set complexity and cost increase
Solution Approach 1:
A universal probe set is designed that can distinguish all four nucleotides (A, T, C, G) using only two unique labels through combinatorial labeling strategies. This multi-functional probe set maintains nucleotide identification accuracy while reducing probe set complexity and the number of required probe types
Solution Approach 2:
The labeling parameters of the probes are changed from using four unique labels (one per nucleotide) to using two unique labels with combinatorial labeling patterns. This parameter change reduces probe set complexity while maintaining the ability to accurately identify all four nucleotide types through the reduced label set
4Measurement precision
If multiple probes are used to distinguish between nucleotides, then nucleotide identification can be achieved, but cost increases
Solution Approach 1:
A universal probe set with two unique labels is designed to identify all four nucleotides, reducing the quantity of different probe types required. This reduces the overall cost of reagents and materials while maintaining nucleotide identification accuracy through the combinatorial labeling approach
Solution Approach 2:
The labeling parameters are changed from four unique labels to two unique labels with combinatorial assignment. This parameter change reduces the cost of probe synthesis, storage, and handling while maintaining the ability to accurately distinguish all nucleotide types through the reduced label set
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 efficiency and reduces costs by allowing multiple bases to be read per cycle, facilitating high-throughput sequencing of large sequences like whole genomes without significant time or cost increases.
Implementation Method 1
If a sequencing probe from the sequencing set has a unique nucleotide that is complementary to the first nucleotide, that sequencing probe hybridizes to the concatemer
Implementation Method 2
The method further includes the step of ligating hybridized sequencing probes to hybridized anchor probes to form ligation products
Data Source
AI summary
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences. In particular, the present invention provides methods and compositions for improving the efficiency of sequencing reactions by using fewer labels to distinguish between nucleotides and by detecting nucleotides at multiple detection positions in a target sequence.