3D Base Calling Using Polony Maps for Accurate Tissue Sequencing
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Solution Overview
Problem
Traditional base calling methods in DNA sequencing struggle with accurate analysis of three-dimensional samples like cells and tissues due to out-of-focus signals and interference from cellular components, leading to inaccurate and unreliable base calling.
Innovation Solution
A system and method for 3D base calling that processes a stack of flow cell images from different axial locations, filtering out-of-focus polonies and background signals to generate accurate image intensities, using a 3D polony map for precise registration and base calling, and performing operations in parallel to speed up the sequencing process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional 2D flow cell images are used for base calling, then the base calling process is simple and fast, but the accuracy is poor due to out-of-focus signals and interference from cellular components in 3D samples
Solution Approach 1:
The patent transitions from 2D flow cell image analysis to 3D volumetric analysis by acquiring images at multiple axial locations (z-levels) and reconstructing three-dimensional polony maps. This dimensional expansion enables the system to resolve out-of-focus signals and distinguish true polony locations from background interference, thereby improving base calling accuracy while managing the increased processing complexity through automated algorithms.
2Reliability
If multiple flow cell images from different axial locations are processed, then out-of-focus polonies can be filtered out, but the computational load and storage requirements increase
Solution Approach 1:
The patent performs preliminary actions by acquiring and storing flow cell images at multiple axial locations during the sequencing process. These pre-acquired images serve as a volumetric dataset that can be processed later to generate 3D polony maps, enabling the filtering of out-of-focus signals and improving reliability without adding significant processing time to the critical base calling path.
Solution Approach 2:
The system creates computational copies of the flow cell image data at different axial levels and processes these copies to generate 3D polony maps. By working with copied and processed versions of the data rather than the original images directly, the system can perform complex 3D analysis while preserving the original data for reference, thereby improving reliability without proportionally increasing processing time.
3Measurement precision
If 3D polony maps are generated from multiple axial locations, then spatial information is retained and out-of-focus signals are removed, but the device complexity and processing requirements increase
Solution Approach 1:
The patent segments the flow cell image data into multiple axial slices or z-levels, processing each layer independently to identify in-focus polonies at specific depths. This segmentation approach transforms the complex 3D processing problem into a series of manageable 2D analysis tasks, improving polony location precision while reducing the overall processing complexity through divide-and-conquer methodology.
Data Source
AI summary
Disclosed herein are system, apparatus, method, and/or computer program product embodiments, and/or combinations and sub-combinations thereof which enables 3D base calling using flow cell images of samples such as in situ cells or tissue to ensure accurate base calling and sequencing analysis of 3D samples. Embodiments of the methods, systems, and media for 3D base calling of flow cell images includes image intensity, location, size, and/or of clusters or polonies to be relied on for accurate base calling.