Densely-packed analyte layers sub-diffraction sequencing
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Solution Overview
Problem
Current sequencing technologies face challenges in achieving high-density analyte packing below the diffraction limit, leading to limitations in resolution and increased costs due to the diffraction limit of optical systems, which hinders the goal of reducing sequencing costs to $10 per 30× human genome.
Innovation Solution
The method involves densely packing analytes on a substrate with a center-to-center spacing below the diffraction limit, using advanced imaging systems and cycled detection to determine precise positions and apply deconvolution techniques for accurate signal resolution, enabling sequencing by synthesis with high accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If analytes are densely packed below the diffraction limit on a substrate, then the data per unit area increases and sequencing cost decreases, but the resolution and signal detection accuracy deteriorate due to optical diffraction limitations
Solution Approach 1:
The patent segments the detection process into multiple cycles, where each cycle detects a subset of analytes with unique barcode sequences. By dividing the dense analyte population into sequentially detected groups, the system achieves high-density packing while maintaining detection accuracy through temporal separation of signals.
Solution Approach 2:
The patent transitions from spatial resolution alone to a combination of spatial and temporal dimensions. Analytes are packed densely in space below the diffraction limit, but are distinguished through multiple detection cycles over time, each targeting specific barcode sequences. This adds the time dimension to resolve signals that are spatially indistinguishable.
2Quantity of substance
If analytes are densely packed on the substrate, then reagent usage decreases and cost reduces, but the ability to resolve individual signals deteriorates due to overlapping optical signals
Solution Approach 1:
The detection process is segmented into multiple cycles, each detecting a specific subset of analytes identified by unique barcode sequences. This segmentation allows dense packing of analytes while resolving individual signals through temporal and sequence-based separation, reducing reagent usage without sacrificing signal resolution.
Solution Approach 2:
The patent introduces barcode sequences as an intermediary layer between the analyte and the detection system. These barcodes serve as unique identifiers that mediate the detection process, allowing the optical system to distinguish between densely packed analytes through sequence-specific binding and temporal detection cycles.
3Device complexity
If standard optical imaging is used for densely packed analytes, then the system complexity remains low, but the sequencing accuracy and read quality deteriorate due to diffraction limit constraints
Solution Approach 1:
The patent segments the sequencing process into multiple detection cycles with cycle-specific barcode sequences. This allows standard optical imaging systems to achieve high sequencing accuracy by detecting analytes in sequential batches rather than attempting to resolve all densely packed analytes simultaneously, avoiding the need for complex super-resolution optics.
Solution Approach 2:
The detection process employs periodic action through multiple cycles, where each cycle targets specific barcode sequences. This periodic detection approach allows standard optical systems to achieve high accuracy by repeatedly imaging the same field with different selective bindings, accumulating signal information over time without requiring complex imaging hardware.
Data Source
AI summary
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These methods and systems may have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.