Oligo-Modified Nucleotide Adapters for Strand-Preserving Library Prep
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Solution Overview
Problem
Existing nucleic acid sequencing library preparation methods involve inefficient and loss-prone adapter addition processes, requiring additional purification steps and leading to sample loss due to incorrect adapter combinations and loss of strandedness information.
Innovation Solution
The use of oligo-modified nucleotide analogues with oligonucleotide adapters that are directly incorporated onto nucleic acids via polymerase-mediated reactions, allowing for efficient 3' adapterization and simplifying workflows, including asymmetric adapter addition without sample loss.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If ligation of adapters onto nucleic acid ends is used, then adapters can be added to nucleic acids, but sample loss occurs due to incorrect adapter combinations and additional purification steps are required
Solution Approach 1:
The adapter is pre-coupled to a nucleotide analogue before the nucleotide is incorporated into the nucleic acid. This preliminary attachment ensures the adapter is already in position and correctly oriented when polymerase incorporates the modified nucleotide, eliminating the need for subsequent ligation steps and preventing sample loss from incorrect adapter combinations.
Solution Approach 2:
The mechanical/chemical ligation process is replaced by polymerase-mediated incorporation. Instead of using ligase to join adapters to nucleic acid ends, the polymerase incorporates the modified nucleotide (with pre-coupled adapter) into the growing nucleic acid chain, substituting a biochemical process for a chemical/mechanical one that is more precise and eliminates purification steps.
2Productivity
If ligation reaction is used to add adapters, then adapters can be incorporated onto nucleic acids, but additional purification steps are necessary to remove undesired ligation products
Solution Approach 1:
The adapter is pre-coupled to the nucleotide analogue in a controlled manner before incorporation. This ensures that when the polymerase incorporates the modified nucleotide, the adapter is already correctly positioned and oriented, producing only the desired product without undesired ligation products that would require purification.
Solution Approach 2:
The ligation reaction and subsequent purification steps are replaced by a single polymerase incorporation step. The polymerase reads the template and incorporates the modified nucleotide with the pre-coupled adapter, creating the final product in one biochemical step without generating byproducts that need removal.
3Adaptability or versatility
If universal adapters are added using conventional methods, then sequencing libraries can be prepared, but strandedness information is lost
Solution Approach 1:
The method introduces local asymmetry by incorporating adapters at specific orientations relative to the nucleic acid strand. By using modified nucleotides with pre-coupled adapters that are incorporated in a strand-specific manner, the adapter orientation reflects the original strand direction, preserving strandedness information while maintaining universal adapter compatibility.
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 increases yield and simplifies sample preparation by reducing steps, enabling PCR-free or PCR-friendly workflows and maintaining strandedness information, while improving library preparation efficiency.
Implementation Method 1
The modified nucleotide can be used to directly incorporate adapters onto nucleic acids as part of sample preparation for downstream processing steps
Implementation Method 2
reacting the 5' oligonucleotide end of the oligonucleotide adapter on the extended nucleic acid with the 3' reactive group to couple the 5' oligonucleotide end to the deoxyribose
Implementation Method 3
cleaving the linker to liberate a 3' end of the oligonucleotide adapter
Data Source
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AI summary
Nucleic acid techniques are disclosed. Embodiments include modified nucleotides 12 with oligonucleotide adapters 24 that are coupled via cleavable linkers 20. Incorporation of the modified nucleotide 12 at a 3' end of a nucleic acid permits end-adapterization via ligation of a free 5' end of the oligonucleotide adapter 24 to a 3' reactive group of the modified nucleotide 12 and cleavage at the cleavable linker 20 to liberate a free 3' end.