Patterned Flow Cell Primer Layout for Simultaneous Paired-End Reads
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Solution Overview
Problem
Existing sequencing technologies face challenges in efficiently performing simultaneous paired-end sequencing due to limitations in primer set configurations and spatial separation of forward and reverse strands, leading to interference in fluorescence signals and suboptimal base calling.
Innovation Solution
The development of a flow cell with a patterned structure featuring distinct primer sets on different regions, utilizing orthogonal cleaving chemistry to spatially separate forward and reverse strands, enabling simultaneous paired-end reads through precise primer attachment and functionalized layers on a multi-layer stack.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If forward and reverse strands are sequenced simultaneously in the same region, then sequencing throughput is improved, but fluorescence signal interference occurs leading to reduced base calling accuracy
Solution Approach 1:
The flow cell surface is segmented into distinct regions: a first region with a first primer set for forward strand sequencing and a second region with a second primer set for reverse strand sequencing. This spatial segmentation allows simultaneous sequencing of both strands without fluorescence signal interference, resolving the contradiction between improved throughput and maintained base calling accuracy.
Solution Approach 2:
The invention transitions from sequential sequencing (one dimension in time) to simultaneous sequencing by adding a spatial dimension. By positioning forward and reverse primer sets in different spatial regions on the flow cell, both strands can be sequenced at the same time without signal interference, achieving both high throughput and high accuracy.
2Device complexity
If primer sets are attached to the same region on the substrate, then device complexity is reduced, but spatial separation of forward and reverse strands cannot be achieved
Solution Approach 1:
The flow cell substrate is divided into multiple functional regions with different primer sets. The first region contains the first primer set while the second region contains the second primer set, providing the necessary spatial separation for simultaneous paired-end sequencing while maintaining a relatively simple overall device structure.
Solution Approach 2:
Different regions of the flow cell are赋予 different local properties through the selective attachment of primer sets. The first region is optimized for forward strand sequencing with its specific primer set, while the second region is optimized for reverse strand sequencing, allowing each region to perform its specific function effectively.
Data Source
Figure 1A~1E
Figure 2A~2D
Figure 3A~3C
AI summary
In one example, a flow cell includes a base support and a protrusion over the base support, where the protrusion is a different material than the base support. The flow cell further includes a first functionalized layer over a first portion of the protrusion, a second functionalized layer over a second portion of the protrusion, and first and second primer sets respectively attached to the first and second functionalized layers.