Flow Cell Primer Attachment via Segmented Hydrogel Domains
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Solution Overview
Problem
Simultaneous paired-end sequencing technologies face challenges in achieving clear signal resolution due to the padlock-like conformation of primer sets on flow cell surfaces, which leads to contamination and interference between signals from different regions.
Innovation Solution
The method involves attaching different primer sets to specific regions of a flow cell using polymeric hydrogels, ensuring orthogonal cleaving chemistry and spatial separation of forward and reverse strands to prevent padlock-like conformation, thereby improving signal resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If primer sets are attached to different regions within each depression using polymeric hydrogels, then simultaneous paired-end sequencing can be performed, but padlock-like conformation occurs causing signal contamination and interference
Solution Approach 1:
The flow cell surface is divided into multiple distinct regions within each depression, with each region containing a specific primer set. The polymeric hydrogel is segmented into discrete domains that spatially separate forward and reverse primer sets, preventing padlock-like conformation and ensuring that signals from different regions remain distinct during simultaneous paired-end sequencing
Solution Approach 2:
Different regions within each depression are赋予 different local properties through selective attachment of specific primer sets to polymeric hydrogel domains. The hydrogel composition or crosslinking density may vary locally to control primer set positioning and orientation, ensuring that each region has the optimal local quality for its specific sequencing function while maintaining spatial separation from other regions
2Loss of time
If different primer sets are attached to different regions, then forward and reverse strands can be sequenced simultaneously, but signal contamination occurs between regions
Solution Approach 1:
The polymeric hydrogel acts as an intermediary medium that physically separates different primer sets into distinct spatial domains within each depression. This hydrogel matrix serves as a mediator that prevents direct interaction between forward and reverse primer sets, thereby eliminating signal contamination while allowing both primer sets to function simultaneously for paired-end sequencing
Solution Approach 2:
The harmful padlock-like conformation is eliminated by extracting or removing the continuous polymeric hydrogel layer and replacing it with segmented, spatially separated hydrogel domains. This extraction of the problematic continuous structure and replacement with discrete separated domains prevents signal contamination between regions
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 signal resolution by reducing or eliminating padlock-like conformation, allowing for simultaneous base calling of both reads without signal contamination, thereby improving the accuracy and clarity of sequencing results.
Implementation Method 1
different primer sets are attached to different regions within each depression and/or on each protrusion of a flow cell surface. These primer sets are attached through polymeric hydrogel(s)
Data Source
AI summary
In an example method, a first functionalized layer is deposited over a resin layer including multi-depth depressions separated by interstitial regions, each depression including a deep portion and a shallow portion adjacent to the deep portion; a photoresist is deposited over the first functionalized layer; an ultraviolet light dosage is directed, through the resin layer, whereby a first photoresist portion generates an insoluble photoresist and a second photoresist portion becomes a soluble photoresist; the soluble photoresist is removed to expose a portion of the first functionalized layer; the portion of the first functionalized layer is removed to expose a portion of the resin layer; a second functionalized layer is deposited over the insoluble photoresist, and over the exposed portion of the resin layer; the insoluble photoresist is removed to expose the first functionalized layer; and the first functionalized layer or the second functionalized layer is removed from the interstitial regions.