Parallel Index and Read Sequencing via Signal Intensity
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Solution Overview
Problem
Next-generation sequencing technologies face limitations in speed due to the need for serial ordering of nucleobase identification in sequencing cycles, which hampers the efficient determination of nucleotide sequences.
Innovation Solution
The method involves hybridizing index and read primers to the template polynucleotide simultaneously, allowing for the simultaneous sequencing of index and insert portions by distinguishing signal intensities from fluorescent emissions, thereby reducing sequencing time and fluidic complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If serial sequencing cycles are performed to identify nucleobases one at a time, then sequencing accuracy is maintained, but sequencing speed is limited
Solution Approach 1:
The template polynucleotide is divided into multiple regions with distinct primer binding sites, allowing multiple primers to bind simultaneously at different locations. This segmentation enables parallel sequencing reactions to occur at multiple sites within the same cluster, transforming the serial identification process into a parallel one and thereby increasing sequencing speed without sacrificing accuracy
Solution Approach 2:
Multiple primer binding sites and their corresponding sequencing reactions are merged into a single cluster on the flow cell. By combining multiple sequencing reactions that occur simultaneously in the same physical space, the system achieves parallel processing of multiple nucleobase identifications, resolving the contradiction between speed and process complexity
2Loss of time
If multiple primers are hybridized to the same cluster for parallel sequencing, then sequencing time is reduced, but signal differentiation becomes more difficult
Solution Approach 1:
Each primer binding site is designed with unique local characteristics, specifically different expected signal intensities based on the number of template strands and primer binding efficiency at each site. This local quality differentiation allows the system to distinguish signals from different primer binding sites within the same cluster, enabling parallel sequencing while maintaining signal detectability and reducing sequencing time
3Productivity
If traditional serial sequencing is performed, then fluidic handling is simplified, but productivity is reduced
Solution Approach 1:
The fluidic system is designed with multi-functionality to handle multiple primer binding sites simultaneously. A single fluidic pathway delivers reagents that can interact with multiple primer binding sites in parallel, and the imaging system captures signals from all sites concurrently. This universal design enables the system to perform multiple sequencing reactions through the same fluidic and imaging infrastructure, increasing productivity without proportionally increasing fluidic system complexity
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 faster sequencing by allowing parallel determination of nucleobase sequences, reducing the time required for sequencing and improving the efficiency of sequencing-by-synthesis workflows.
Implementation Method 1
deoxyribonucleic acid analogs conjugated with fluorescent labels may be hybridized to the target polynucleotides at a certain site, and excitation light sources may be used to excite the fluorescent labels on the deoxyribonucleic acid analogs. Detectors may capture fluorescent emissions from the fluorescent labels
Data Source
AI summary
Systems and methods of identifying nucleobases in a template polynucleotide are disclosed. In one embodiment, such a method may include providing a substrate comprising a plurality of the template polynucleotides in a cluster. The method may further include generating light to stimulate fluorescent emissions from the cluster. The method may further include receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a first site. The method may further include receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a second site. The method may further include identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.