Double-Stranded Splint Adaptors for Indexed Circular DNA Library Prep
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for preparing covalently closed circular DNA libraries are inefficient and lack the ability to incorporate unique index sequences, which are essential for high-throughput sequencing and pooling of multiple libraries.
Innovation Solution
The use of double-stranded splint adaptors that hybridize with single-stranded library molecules to form library-splint complexes, which are then circularized and ligated to create covalently closed circular molecules, allowing for the incorporation of universal and unique sequences for sequencing workflows.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used for preparing circular DNA libraries, then the process is simpler, but the efficiency and ability to incorporate unique index sequences are insufficient
Solution Approach 1:
The splint adaptor is divided into multiple functional regions: a first region complementary to the library molecule end, a second region containing universal adaptor sequences, and a third region with unique index sequences. This segmentation allows each region to perform its specific function independently while contributing to the overall efficiency of library preparation and sequencing throughput
Solution Approach 2:
The splint adaptor serves multiple functions simultaneously: it acts as a hybridization probe for circularization, provides universal adaptor sequences for downstream processing, and incorporates unique index sequences for sample identification. This multi-functionality increases sequencing throughput without proportionally increasing process complexity
2Reliability
If double-stranded splint adaptors are used to circularize library molecules, then unique index sequences can be incorporated, but the hybridization process becomes more complex
Solution Approach 1:
Different regions of the splint adaptor have specialized sequences optimized for their specific functions: the first region is designed for complementary hybridization to library ends, the second region contains universal adaptor sequences for reliable downstream processing, and the third region provides unique index sequences. This local optimization ensures high circularization efficiency while managing structural complexity through functional specialization
Solution Approach 2:
The double-stranded splint adaptor acts as an intermediary molecule that mediates the circularization of linear library molecules. By providing a structured template with complementary regions, it facilitates reliable hybridization and circularization while the universal and unique sequences serve as intermediaries for subsequent sequencing workflows
3Adaptability or versatility
If traditional library preparation methods are used, then the process is faster, but the ability to support high-throughput sequencing and pooling is limited
Solution Approach 1:
The splint adaptor incorporates universal adaptor sequences that are compatible with standard downstream sequencing workflows, while simultaneously including unique index sequences for sample identification and pooling. This universal design enables the method to adapt to high-throughput sequencing requirements and increase data output without sacrificing workflow compatibility
4Productivity
If unique index sequences are added during library preparation, then pooling and simultaneous sequencing are enabled, but the library preparation process becomes more complex
Solution Approach 1:
The unique index sequences are merged into the splint adaptor structure itself, rather than being added as separate components. This integration allows unique identifiers to be incorporated during the circularization step, enabling pooling and simultaneous sequencing while simplifying the overall preparation process by combining multiple functions into a single reagent
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 method enables efficient preparation of circular library molecules with unique index sequences, compatible with next-generation sequencing technologies, enhancing sequencing throughput and data output.
Implementation Method 1
the first splint strand comprises a first region (320), an internal region (310), and a second region (330); wherein the internal region of the first splint strand (310) is hybridized to the second splint strand (400), wherein the first region of the first splint strand (320) is hybridized to the at least first left universal adaptor sequence (120) of the library molecule, and wherein a second region of the first splint strand (330) is hybridized to the at least first right universal sequence (130) of the library molecule
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. 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, an index sequence and/or a random sequence.