Parallel Sequencing via Staggered Fragment Assembly
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Solution Overview
Problem
Current DNA sequencing technologies face challenges in achieving rapid, high-throughput, and cost-effective sequencing, particularly for applications like point-of-care pathogen detection and multiplex nucleic acid analysis, which require efficient discrimination of single-base differences at multiple loci and are hindered by complex hybridization conditions and limited multiplexing capabilities.
Innovation Solution
The method involves using a plurality of substrates with capture probes complementary to a target nucleic acid, where each substrate undergoes sequential extension cycles with native and labeled dNTPs, allowing for staggered fragment generation and assembly of sequence reads to determine the nucleotide sequence of the target nucleic acid, enabling parallel sequencing and reducing sequencing time and cost.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If traditional DNA sequencing methods are used, then sequencing accuracy is maintained, but sequencing speed and throughput are limited
Solution Approach 1:
The invention divides the sequencing process into multiple parallel reactions, each occurring on separate substrates. Multiple capture probes are immobilized on each substrate, and each probe captures different target sequences simultaneously. This segmentation allows numerous sequencing reactions to proceed in parallel, dramatically increasing throughput while maintaining accuracy through independent reaction channels.
Solution Approach 2:
The invention transitions from linear sequential sequencing to parallel processing by utilizing multiple substrates and multiple capture probes per substrate. This dimensional expansion from single-reaction sequencing to multi-reaction parallel sequencing enables simultaneous processing of numerous target sequences, effectively increasing sequencing speed and throughput without sacrificing accuracy.
2Measurement precision
If complex hybridization conditions are used to achieve single-base discrimination, then detection precision is improved, but device complexity and operational difficulty increase
Solution Approach 1:
The invention employs capture probes with locally optimized sequences designed to discriminate single-base differences at specific target loci. Each capture probe is engineered with precise sequence characteristics tailored to its specific target, enabling high-resolution single-base discrimination without requiring complex global hybridization conditions. This local optimization approach maintains detection precision while simplifying overall system operation.
3Productivity
If traditional sequencing methods are used, then cost-effectiveness is maintained for small-scale applications, but productivity and scalability worsen
Solution Approach 1:
The invention combines multiple sequencing reactions into a single integrated system using solid substrates with immobilized capture probes. By merging numerous parallel reactions onto unified substrates and using universal detection reagents, the system achieves high throughput while reducing per-reaction costs. This consolidation eliminates the need for separate processing of individual reactions, improving productivity and cost-effectiveness for large-scale sequencing applications.
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 rapid and cost-effective sequencing of large genomic regions, accommodating low-efficiency chemistry and sequencing very long fragments, while enhancing multiplexing capabilities and reducing the time required for sequencing, making it suitable for applications such as pathogen identification and genetic disease diagnosis.
Implementation Method 1
forming a hybridization complex on each of the different substrates (i), wherein the hybridization complex comprises the capture probe and the target nucleic acid, wherein the capture probe is hybridized to the portion of the target nucleic acid
Implementation Method 2
extending the capture probe in the hybridization complex on each of the different substrates (i) by repeating i-1 times the step of: contacting the hybridization complex on each of the different substrates (i) sequentially with 1) one of native dATP, dCTP, dGTP and dTTP; or 2) a mixture of two or three native dNTPs followed by one or more rounds of two or more different native dNTPs until all four native dNTPs are added at least once, in the presence of a polymerase, thereby extending the capture probe in the hybridization complex by one or more bases
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3~4B
AI summary
The present invention provides methods for native extension parallel sequencing of polynucleotide.