Flow Cell Regional Cleavage Chemistry for Paired-End Signal Separation
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Solution Overview
Problem
Existing sequencing technologies face challenges in efficiently and simultaneously sequencing both forward and reverse strands, leading to overlapping fluorescence signals and difficulties in base calling.
Innovation Solution
The development of flow cells with orthogonal cleaving chemistry in different regions, allowing for spatial separation of forward and reverse strands, enabling simultaneous paired-end sequencing by using distinct primer sets or identical cleavage sites in different primers, thereby separating fluorescence signals and facilitating simultaneous base calling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If sequential paired-end sequencing is used, then forward strands are sequenced and removed before reverse strands are sequenced, but this approach increases sequencing time and reduces productivity
Solution Approach 1:
The flow cell surface is segmented into multiple regions with different primer sets attached to different regions. This allows forward and reverse strands to be sequenced simultaneously in different spatial locations rather than sequentially, thereby increasing throughput without increasing sequencing time
Solution Approach 2:
The patent transitions from temporal sequencing (sequential processing of forward then reverse strands) to spatial sequencing (simultaneous processing in different regions). By adding a spatial dimension to the sequencing process, both strands can be processed in parallel, resolving the time-productivity contradiction
2Productivity
If simultaneous paired-end sequencing is used, then forward and reverse strands are sequenced at the same time, but this causes overlapping fluorescence signals that reduce measurement precision
Solution Approach 1:
Different regions of the flow cell are assigned different primer sets with distinct fluorescent labeling schemes. This local differentiation ensures that fluorescence signals from forward and reverse strand sequencing originate from different spatial locations and use different color codes, preventing signal overlap and maintaining measurement precision
Solution Approach 2:
The patent employs different fluorescent colors to label nucleotides in forward versus reverse strand sequencing. By changing the color dimension of the signal, the system can distinguish between forward and reverse strand incorporation events even when they occur simultaneously, thereby maintaining base calling accuracy while enabling parallel sequencing
3Measurement precision
If orthogonal cleaving chemistry is implemented in different regions, then spatial separation of forward and reverse strands is achieved, but this increases device complexity
Solution Approach 1:
The flow cell surface uses a universal functionalized layer chemistry that can bind multiple different primer sets. This multi-functional surface allows different regions to be programmed with different primers while maintaining a consistent underlying chemical architecture, thereby achieving signal separation without proportionally increasing structural complexity
Solution Approach 2:
The patent uses identical or similar functionalized layer compositions across different regions, copying the successful chemistry design and varying only the attached primer sequences. This approach achieves regional differentiation through simple molecular variation rather than complex structural changes, minimizing the increase in device complexity
Data Source
AI summary
In an example of a method for making a flow cell, a metal material is sputtered over a transparent substrate including depressions separated by interstitial regions to form a metal film having a first thickness over the interstitial regions and having a second thickness over the depressions, the second thickness being about 30 nm or less and being at least ⅓ times smaller than the first thickness. A light sensitive material is deposited over the metal film; and the metal film is used to develop the light sensitive material through the transparent substrate to define an altered light sensitive material at a first predetermined region over the transparent substrate. The altered light sensitive material is utilized to generate a functionalized layer at the first predetermined region or at a second predetermined region over the transparent substrate.