Template Strand Concatemerization for Brighter Sequencing Clusters
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in reducing costs and improving efficiency, particularly in library generation, clonal amplification, sequencing biochemistry, imaging technology, and sequence assembly from short reads, which limit the cost-effectiveness and quality of genome analysis.
Innovation Solution
The method involves surface amplification by concatenating template strands during the bridge amplification process, allowing multiple copies of the template strand to be extended onto the flowcell surface, resulting in brighter clusters that support longer read lengths, better read quality, and increased system robustness without altering the density of standard cluster amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If standard cluster amplification is used, then sequencing can be performed, but the clusters are not bright enough to support longer read lengths and higher quality reads
Solution Approach 1:
The patent merges multiple template strands onto a single flowcell surface primer through concatemerization, creating clusters with multiple copies of the template strand. This merging approach increases the number of fluorescent signals per cluster, thereby increasing cluster brightness while maintaining the same spatial density, which directly addresses the contradiction between brightness and read quality/length
Solution Approach 2:
The patent changes the parameter of template strand copy number per cluster by implementing concatemerization during bridge amplification. By increasing the copy number of template strands attached to each flowcell surface primer, the cluster brightness is enhanced without changing the physical density of clusters on the flowcell, thus improving read quality and supported read length
2Ease of operation
If sequencing is performed on single molecules, then cyclic sequencing can occur asynchronously, but the method lacks the signal amplification needed for robust sequencing
Solution Approach 1:
The patent combines the advantages of single-molecule sequencing with cluster amplification by attaching multiple copies of the template strand to each flowcell surface primer. This allows asynchronous cyclic sequencing to occur while providing sufficient signal amplification through the concatemerized template strands, thereby resolving the contradiction between ease of operation and sequencing robustness
3Quantity of substance
If clonal amplification is performed with high density, then cost-effective sequencing is achieved, but the background noise grows with each sequencing cycle limiting read length
Solution Approach 1:
The patent changes the parameter of template strand copy number per cluster through concatemerization, increasing the signal intensity from multiple template copies. This parameter change allows maintaining high cluster density for cost-effective sequencing while the enhanced signal from concatemerized templates counteracts the background noise accumulation, enabling longer read lengths
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 the quality and length of sequencing reads, reduces costs, and improves the robustness of the sequencing process, enabling more efficient and cost-effective genome analysis.
Implementation Method 1
applying the target nucleic acid to the solid support under conditions suitable for hybridization whereby the first region of known sequence hybridizes to the forward amplification primer
Implementation Method 2
extending the hybridized forward amplification primer to generate an immobilized template comprising a complementary copy of the target nucleic acid
Data Source
AI summary
Presented herein are methods and compositions for concatenating template strands during the bridge amplification process. The methods are useful for surface amplification at improved densities. The methods and compositions provided herein enable creation of clusters that are brighter, but at the same densities as currently achieved using standard cluster amplification.