Double-Stranded Splint Adaptors for PCR-Free Circular DNA Libraries
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Solution Overview
Problem
Next-generation sequencing methods face challenges with short sequencing reads that require computational reassembly, which is time-consuming, laborious, and computationally demanding, especially for complex genomes, often failing to recover crucial haplotype information.
Innovation Solution
The use of double-stranded splint adaptors that hybridize with single-stranded nucleic acid library molecules to form library-splint complexes, which are then circularized and ligated to create covalently closed circular molecules, facilitating downstream amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If next-generation sequencing methods are used, then sequencing cost is reduced, but sequencing read length becomes short requiring computational reassembly
Solution Approach 1:
The patent performs preliminary circularization of library molecules before sequencing, creating covalently closed circular DNA structures that enable long-read sequencing without requiring subsequent computational reassembly. This preliminary structural preparation eliminates the need for complex post-sequencing assembly algorithms while maintaining cost-effectiveness.
Solution Approach 2:
The patent introduces circularized library molecules as an intermediary form between traditional linear DNA and sequencing output. This circular intermediate structure allows direct long-read sequencing that bypasses the need for computational reassembly, acting as a mediator that transforms the sequencing workflow.
2Loss of information
If computational reassembly of short reads is performed, then full sequences can be obtained, but time and computational resources are significantly consumed
Solution Approach 1:
The patent performs preliminary circularization of library molecules before sequencing, creating covalently closed circular DNA structures that enable long-read sequencing without requiring subsequent computational reassembly. This preliminary structural preparation eliminates the need for complex post-sequencing assembly algorithms while maintaining cost-effectiveness.
3Productivity
If traditional library preparation methods are used, then standard sequencing workflows are maintained, but PCR artifacts and biases are introduced
Solution Approach 1:
The patent extracts and removes the PCR amplification step from the traditional library preparation workflow. By using circularized library molecules that can be directly sequenced without PCR, the method eliminates PCR artifacts and biases while maintaining workflow efficiency through streamlined preparation procedures.
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 the time and computational requirements for sequencing, enabling efficient assembly and recovery of accurate sequences, particularly for complex genomes.
Implementation Method 1
The double-stranded splint adaptors can hybridize to portions of library molecules to form library-splint complexes
Implementation Method 2
the nicks can be ligated to form covalently closed circular molecules
Data Source
AI summary
The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors, e.g., PCR-free workflows. The double-stranded splint adaptors (200) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor (200) comprises a first splint strand (long splint strand (300)) and a second splint strand (short splint strand (400)), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor (200) having a double-stranded region and two flanking single-stranded regions. The second splint strand (400) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.