Super-Resolution Sequencing with Reversible Terminators
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Solution Overview
Problem
Existing next-generation sequencing (NGS) technologies face challenges such as high reagent consumption, large instrument size, reagent waste, high error rates, low throughput, and difficulties in sequencing homopolymer regions due to asynchronous noise and diffusible signals, particularly in methods like Illumina's reversible terminator approach and Pacific Biosciences' real-time sequencing.
Innovation Solution
A super-resolution sequencing method utilizing template-derived chain extension with detectable labeled nucleotides, enabling controlled incorporation of nucleotides one at a time within a closed system, and employing super-resolution imaging techniques like PAINT and STORM to localize and identify nucleotides with high precision, allowing parallel sequencing of multiple templates in a smaller footprint.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reversible terminator approach with syringe pump reagent delivery is used, then sequencing can be conducted on clusters of target polynucleotides, but large volume of reagents is consumed and instrument size becomes large
Solution Approach 1:
The invention segments the sequencing process into two distinct phases: a loading phase where templates are immobilized on the flow cell surface, and a sequencing phase where the same templates are sequenced using a different reagent delivery mechanism. This segmentation allows optimization of reagent usage during sequencing by eliminating continuous reagent flow and wash steps, thereby reducing overall reagent consumption while maintaining high throughput
Solution Approach 2:
The invention introduces an intermediary mechanism - a flow cell with integrated microfluidic channels that acts as a mediator between the reagents and the templates. This intermediary structure enables precise control of reagent delivery directly to the template locations, eliminating the need for large-volume syringe pump delivery and extensive wash steps, thus reducing reagent consumption while maintaining sequencing productivity
2Productivity
If reversible terminator approach with syringe pump reagent delivery is used, then sequencing can be conducted on clusters of target polynucleotides, but instrument size becomes large
Solution Approach 1:
The invention segments the instrument into modular components: a compact flow cell unit with integrated microfluidic channels, a simplified reagent delivery system, and a detection system. This modular segmentation allows the instrument to achieve high sequencing throughput through parallel processing of multiple templates on the flow cell while maintaining a compact overall size by eliminating large syringe pumps and extensive fluid handling mechanisms
Solution Approach 2:
The invention transitions from a three-dimensional bulk reagent delivery system (syringe pumps delivering reagents through large-volume fluid paths) to a two-dimensional surface-based approach where templates are immobilized on a planar flow cell surface and reagents are delivered through integrated microchannels. This dimensional change enables high throughput sequencing of multiple templates in parallel while significantly reducing the instrument's footprint
3Loss of substance
If real-time sequencing with terminal phosphate label is used, then reagents can be loaded at start without reagent exchange, but error rate becomes high
Solution Approach 1:
The invention segments the nucleotide incorporation process into controlled discrete steps using reversible terminators, where only one type of nucleotide is incorporated at a time. This segmentation allows for precise detection and confirmation of each incorporation event, significantly reducing error rates compared to continuous real-time sequencing, while the flow cell architecture enables efficient reagent usage through direct delivery to immobilized templates
Solution Approach 2:
The invention changes the chemical parameters of the nucleotides by incorporating reversible terminator groups that allow controlled single-nucleotide incorporation. This parameter change enables the system to achieve both low error rates (through controlled stepwise incorporation and detection) and reduced reagent consumption (through the flow cell's efficient reagent delivery and lack of extensive wash steps)
4Loss of time
If continuous illumination for real-time sequencing is used, then nucleotide incorporation can be monitored in real-time, but light-induced damage to complex occurs and throughput is low
Solution Approach 1:
The invention employs periodic illumination rather than continuous illumination, where light is applied only during specific detection windows when nucleotide incorporation events are being monitored. This periodic action reduces light-induced damage to the templates and reagents while maintaining the ability to sequence rapidly through the flow cell's efficient architecture and parallel processing capability
Solution Approach 2:
The invention converts the potential harm of light exposure by using non-invasive or minimally invasive labeling strategies (such as fluorescent labels on terminators that are removed after detection) and by optimizing illumination timing to occur only when necessary for detection. This approach maintains rapid sequencing throughput while minimizing photodamage, effectively turning the potential harmful effect into a beneficial detection mechanism
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 reduces reagent consumption, minimizes instrument size, and achieves high accuracy and throughput by sequencing densely packed molecules with reduced error rates, overcoming the limitations of current NGS technologies.
Implementation Method 1
template-derived chain extension, where a sequencing cycle comprises determination of a single nucleotide in the growing chain
Implementation Method 2
employing super-resolution imaging techniques like PAINT and STORM to localize and identify nucleotides with high precision
Data Source
Figure 5
Figure 6
Figure 7A~7B
AI summary
A method for template-directed sequencing-by- synthesis of an array of target polynucleotide can include: (a) providing an array of target polynucleotides in a fluidic vessel; (b) contacting the array of polynucleotides with a solution comprising (i) polymerization complex and (ii) reversibly terminating and differently labeled A,C,G, and T/U nucleotides; (c) incorporating one of the differently labeled nucleotides, using the polymerization complex, into a chain complementary to at least one of the array of polynucleotides; (d) binding imaging tags to the differently labeled nucleotides of step (c); (e) imaging and storing the identity and position of the imaging tags of step (d); (f) reversing termination (b)-(e); (g) repeating steps (b)-(e) and assembling a sequence for each of the array of target polynucleotides from the stored identity and position of the imaging tags, optionally as a homogeneous or one pot reaction. Additional methods of sequencing target polynucleotides are described herein.