Double-Strand Sequencing via Inter-Strand Cross-Linking
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Solution Overview
Problem
Existing methods for sequencing double stranded nucleic acids face challenges in accurately sequencing both strands without using hairpin adapters, which can increase complexity, sample preparation time, and risk contamination, while conventional techniques often discard information from the complementary strand.
Innovation Solution
Forming an inter-strand cross-link between the strands of a double stranded nucleic acid using a cross-linking agent, followed by sequencing the cross-linked construct with a single molecule technique, allowing enzymes to process both strands sequentially.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If hairpin adapters are used to sequence both strands of double stranded nucleic acid, then both strands can be sequenced, but device complexity and sample preparation time increase
Solution Approach 1:
The invention extracts and removes the hairpin adapter component from the sequencing system. Instead of using hairpin adapters to link strands, the method sequences each strand independently after denaturation, eliminating the adapter-related complexity while still achieving comprehensive sequencing of both strands.
Solution Approach 2:
The sequencing process is segmented into independent steps: denaturation of double-stranded nucleic acid into separate single strands, followed by independent sequencing of each strand. This segmentation removes the need for complex adapter-based linking while ensuring both strands are sequenced.
2Loss of information
If hairpin adapters are used to sequence both strands, then both strands can be sequenced, but sample preparation time increases
Solution Approach 1:
The hairpin adapter attachment step is extracted and removed from the sample preparation workflow. The method directly sequences denatured strands without requiring adapter ligation, significantly reducing preparation time while maintaining complete sequencing coverage.
Solution Approach 2:
The denaturation step is performed as a preliminary action before sequencing, separating the strands in advance. This allows each strand to be sequenced independently without requiring time-consuming adapter attachment, streamlining the overall process.
3Loss of information
If hairpin adapters are used to sequence both strands, then both strands can be sequenced, but risk of contamination increases
Solution Approach 1:
The hairpin adapter component is removed from the system, eliminating the contamination risk associated with adapter attachment and handling. The method achieves dual-strand sequencing through independent processing of denatured strands, reducing potential contamination points.
4Ease of manufacture
If conventional sequencing methods are used, then sequencing can be performed, but information from complementary strand is discarded
Solution Approach 1:
The sequencing process is divided into independent sequencing reactions for each strand. After denaturation separates the double-stranded nucleic acid into individual strands, each strand undergoes sequencing independently, ensuring that information from both strands is captured without requiring complex adapter-based methods.
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
Provides orthogonal proof-reading sequence information and reduces the need for hairpin adapters, simplifying the process and minimizing contamination risks while enhancing sequencing accuracy.
Implementation Method 1
forming an inter-strand cross-link between the first and second strands of the target double stranded nucleic acid by exposing the target double stranded nucleic acid to a cross-linking agent
Data Source
Figure 1A~1D
Figure 2(a)~2(c)
Figure 3A~3C
AI summary
Provided herein is a method of sequencing a target double stranded nucleic acid. The method comprises contacting the double stranded nucleic acid with a reagent as described herein to form a construct and sequencing the construct using a single-molecule sequencing technique as described herein. Associated products and kits are further provided.