Single-Cell Sequencing with Localized Duplex Strand Pairing
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Solution Overview
Problem
Existing nucleic acid sequencing methods, particularly sequencing-by-synthesis (SBS), are costly and labor-intensive due to the need for deactivation and removal of fluorescent signals, limiting their application in high-throughput sequencing, especially in scenarios like COVID-19 diagnostics where rapid sequencing of large numbers of samples is required.
Innovation Solution
A method involving localizing double-stranded nucleic acids to a substrate, generating single-stranded nucleic acids, restricting their diffusion, and attaching them near the location for sequencing reads, which allows for error reduction and efficient sequencing without complete photobleaching of fluorescent signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cycling deactivation and removal of fluorescent labels is performed, then sequencing accuracy is maintained, but sequencing cost and time increase significantly
Solution Approach 1:
The patent extracts and removes the problematic cycling deactivation and removal steps from the sequencing process. By using a non-cyclic method where fluorescent labels are incorporated and detected without requiring subsequent deactivation or removal, the patent eliminates the time-consuming wash and deactivation cycles while maintaining sequencing accuracy through direct signal detection and molecular barcode identification.
Solution Approach 2:
The patent implements continuous nucleotide incorporation and signal detection without interruption for deactivation or removal. The fluorescent labels remain on the nucleic acid molecules throughout the sequencing process, allowing continuous sequencing reads to be obtained from multiple molecules simultaneously without the cyclic interruptions required in traditional methods.
2Measurement precision
If cycling deactivation and removal of fluorescent labels is performed, then sequencing accuracy is maintained, but device complexity and cost increase
Solution Approach 1:
The patent removes the complex cycling deactivation and removal mechanisms from the sequencing system. By eliminating the need for fluorescent label removal steps, the patent simplifies the device architecture, reducing the number of required reagent delivery systems, wash mechanisms, and signal deactivation controls while maintaining accurate sequencing through direct fluorescent signal detection.
Solution Approach 2:
The patent employs disposable fluorescent labels that are incorporated into the nucleic acid molecules and remain there throughout the sequencing process. These single-use fluorescent markers eliminate the need for expensive, complex fluorescent label removal systems and deactivation mechanisms, reducing overall device complexity and operational cost.
3Object-generated harmful factors
If complete photobleaching of fluorescent signals is performed, then signal interference is reduced, but photo-damage to nucleic acids increases
Solution Approach 1:
The patent extracts and eliminates the photobleaching step from the sequencing process. By using fluorescent labels that do not require deactivation or removal, the patent avoids the harmful effects of complete photobleaching while maintaining adequate signal detection through controlled fluorescence imaging and molecular barcode identification.
Solution Approach 2:
The patent converts the potential harm of fluorescent signal persistence into a benefit by utilizing the continuous fluorescent signal for multiple sequencing reads. Rather than requiring signal removal to prevent interference, the patent employs molecular barcodes and sequential detection methods to distinguish between different nucleic acid molecules, turning the persistent fluorescent signal into a useful feature for high-throughput sequencing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach reduces sequencing costs and enhances throughput by allowing continuous nucleotide incorporation and real-time sequencing, minimizing photo-damage and signal interference, thereby improving sequencing accuracy and efficiency.
Implementation Method 1
restricting diffusion of the first and/or second single stranded nucleic acids
Data Source
AI summary
The present disclosure in some aspects relates to real-time nucleic acid sequencing, including real-time sequencing, single molecule sequencing, long-read sequencing, and/or single-cell sequencing. Also described herein are methods of error reduction and of analyzing sequencing data obtained from the sequencing methods. In one aspect, provided herein is a method for nucleic acid sequencing, comprising: a) localizing a double stranded nucleic acid to a location on a substrate; b) generating a first single stranded nucleic acid and a second single stranded nucleic acid from the localized double stranded nucleic acid; c) restricting diffusion of the first and/or second single stranded nucleic acids; d) attaching the first and second single stranded nucleic acids at sites near the location on the substrate; e) obtaining sequencing reads from the attached first single stranded nucleic acid and sequencing reads from the attached second single stranded nucleic acid. In some embodiments, the method further comprises: f) associating a sequence of the first single stranded nucleic acid with a sequence of the second single stranded nucleic acid, thereby determining a sequence of the double stranded nucleic acid.