Nucleic Acid Library Preparation via Single-Stranded Ligation
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Solution Overview
Problem
Current methods for preparing nucleic acid libraries for next-generation sequencing are labor-intensive and require multiple hands-on steps, often involving complex processes like transposome systems for simultaneous fragmentation and adaptor ligation, which can be inefficient and prone to errors.
Innovation Solution
A method involving the ligation of single-stranded nucleic acids to adaptors, where the 3' ends of the first adaptor have a blocking group to prevent self-concatemerization, followed by phosphorylation and ligation of a second adaptor to the 5' ends, using a single-stranded ligase in the presence of a volume excluding agent like PEG, to efficiently prepare nucleic acid libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If transposome systems are used for simultaneous genomic fragmentation and adaptor ligation, then the library preparation process is simplified, but the method becomes prone to errors and less efficient
Solution Approach 1:
The patent divides the library preparation process into distinct sequential steps: fragmentation, end repair, adaptor ligation, and phosphorylation. By separating these operations that were previously combined in transposome systems, the method achieves better control over each step, reducing errors while maintaining process efficiency.
Solution Approach 2:
The patent performs end repair and phosphorylation of nucleic acid fragments before adaptor ligation. This preliminary preparation ensures that all fragments are properly processed and phosphorylated in advance, preventing errors during the ligation step and improving overall reliability.
2Manufacturing precision
If multiple hands-on steps are performed at different stages, then the library preparation can be completed, but the process becomes labor-intensive and time-consuming
Solution Approach 1:
The patent combines multiple operations into unified reaction steps. For example, end repair and phosphorylation are performed in the same reaction vessel using a single enzyme mix, and adaptor ligation is performed in the same volume without intermediate purification steps, significantly reducing hands-on time while maintaining completeness.
Solution Approach 2:
The patent maintains continuous reaction conditions throughout the library preparation process. Reactions proceed sequentially in the same volume without interruption or transfer between vessels, eliminating idle time and reducing manual intervention while ensuring complete processing of all samples.
3Device complexity
If adaptors are ligated without blocking groups, then the ligation process is simpler, but self-concatemerization occurs increasing primer-dimer formation
Solution Approach 1:
The patent introduces blocking groups at specific local positions on the adaptor molecules (at the 3' end of the forward adaptor and 5' end of the reverse adaptor). This localized modification prevents self-concatemerization without affecting the overall adaptor structure or its binding functionality, effectively eliminating primer-dimer formation.
4Loss of time
If dephosphorylation of 5' ends is not performed, then the process is shorter, but self-concatemerization of nucleic acids occurs
Solution Approach 1:
The patent performs dephosphorylation of the 5' ends of nucleic acid fragments as a preliminary step before adaptor ligation. By removing phosphate groups in advance, the method prevents self-concatemerization during the ligation reaction, ensuring that only proper adaptor-nucleic acid ligation occurs without time loss during or after the main reaction.
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 likelihood of primer-dimer formation and allows for the preparation of nucleic acid libraries in a single reaction volume, enhancing the efficiency and accuracy of library preparation by inhibiting self-concatemerization and facilitating the incorporation of indexes for molecular identification.
Implementation Method 1
ligating a first adaptor to the 3' ends of the single-stranded nucleic acids in the presence of a ligase
Implementation Method 2
wherein the 3' end of the first adaptor comprises a blocking group
Implementation Method 3
phosphorylating the 5' ends of the ligated single-stranded nucleic acids
Implementation Method 4
using a single-stranded ligase in the presence of a volume excluding agent like PEG
Data Source
AI summary
Systems, methods and compositions provided herein relate to the preparation of nucleic acid libraries. Some embodiments include the preparation of nucleic acid libraries by ligation of single-stranded nucleic acids.