3D Flow Cell Imaging for High-Density Sequencing Base Calling
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Solution Overview
Problem
Traditional 3D sequencing systems face limitations in generating reliable sequencing results due to spatially overlapped clusters or polonies, leading to reduced sequencing throughput and increased time and effort in hybridizing different primers.
Innovation Solution
A system and method for 3D sequencing that acquires a stack of flow cell images at multiple z-levels, registers these images to a common coordinate system, and performs accurate base calling, allowing higher spatial density sequencing without additional primers or system upgrades.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional 3D sequencing is used with spatially separated clusters, then reliable sequencing results can be obtained, but sequencing throughput is limited
Solution Approach 1:
The patent transitions from traditional 2D flow cell sequencing to 3D flow cell sequencing, utilizing the third dimension (depth/z-axis) to accommodate additional clusters. This dimensional expansion allows significantly more clusters to be sequenced simultaneously while maintaining spatial separation through depth, thereby increasing throughput without sacrificing reliability
Solution Approach 2:
The patent segments the sequencing space into multiple focal planes or z-levels within the 3D flow cell. Each plane can be independently imaged and processed, allowing clusters at different depths to be sequentially or simultaneously analyzed. This segmentation enables high-density clustering while maintaining the ability to resolve individual clusters through focal plane separation
2Adaptability or versatility
If different sequencing primers are used to sequence different clusters, then comprehensive sequencing can be achieved, but additional time and effort are required for hybridization and blocking
Solution Approach 1:
The patent employs universal primers that can bind to and sequence all clusters regardless of their spatial location or depth within the 3D flow cell. This eliminates the need for different primers for different clusters, reducing hybridization steps and time while maintaining comprehensive sequencing coverage through the versatility of the universal primer design
Solution Approach 2:
The patent enables continuous sequencing operations by eliminating the need to block and unblock different cluster groups sequentially. Universal primers allow all clusters to be sequenced in a continuous manner without interruption for hybridization/rehybridization cycles, thereby reducing total sequencing time while maintaining adaptability
Data Source
AI summary
Described herein are aspects for sequencing three-dimensional samples using flow cell images. An aspect begins by obtaining a plurality of subsets of flow cell images of a sample in a plurality of sequencing cycles from a subset of channels. The aspect then generates base calls for the sample based on the subsets of flow cell images.