Double-Stranded Splint Adaptors for Indexed Circular Library Preparation
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Solution Overview
Problem
Current methods for preparing libraries of covalently closed circular molecules are inefficient and do not effectively incorporate unique index sequences, limiting the compatibility with next-generation sequencing technologies that require high throughput and simultaneous sequencing of multiple libraries.
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 ligated to create covalently closed circular molecules, allowing for downstream amplification and sequencing workflows.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional library preparation methods are used, then the process is simpler, but the efficiency and ability to incorporate unique index sequences is poor
Solution Approach 1:
The library preparation process is segmented into distinct functional modules: (i) library molecules with universal adaptor sequences, (ii) double-stranded splint adaptors with internal regions for hybridization, and (iii) indexed splint adaptors with unique index sequences. This segmentation allows each component to perform its specific function efficiently while enabling high-throughput sequencing through modular assembly and pooling of multiple libraries.
2Adaptability or versatility
If unique index sequences are added to DNA fragments, then multiple libraries can be pooled and sequenced simultaneously, but the preparation process becomes more complex
Solution Approach 1:
The splint adaptor design incorporates universal binding regions that can hybridize to universal adaptor sequences on library molecules, while simultaneously accommodating unique index sequences. This multi-functionality allows the same basic adaptor structure to serve multiple libraries with different indices, enabling multiplexing without requiring fundamentally different preparation protocols for each library.
Solution Approach 2:
Unique index sequences are incorporated into the splint adaptors during the library preparation stage, before the actual sequencing process. This preliminary incorporation of indices allows multiple libraries to be prepared and pooled in advance, and then sequenced simultaneously in a single run, rather than requiring separate sequencing runs for each library.
3Reliability
If double-stranded splint adaptors are used to form circular molecules, then compatibility with next-generation sequencing is improved, but the ligation process requires precise conditions
Solution Approach 1:
The double-stranded splint adaptors act as intermediary molecules that bridge the linear library molecules and the desired circular structure. The splint adaptors hybridize to complementary sequences on the library molecules and provide properly positioned ends for ligation, facilitating efficient circularization. This intermediary role ensures reliable formation of circular molecules that are compatible with next-generation sequencing workflows.
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 enables efficient preparation of circular library molecules with unique index sequences, enhancing compatibility with next-generation sequencing technologies and enabling high-throughput sequencing of pooled libraries.
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.