Targeted Nucleic Acid Library Preparation with Cleavable Adaptors
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Solution Overview
Problem
Existing methods for preparing nucleic acid libraries are inefficient and do not allow for rapid production of highly multiplexed targeted libraries, particularly in complex samples, and often result in primer dimers that hinder accurate analysis.
Innovation Solution
A method involving the use of nucleic acid adaptors with universal handle sequences and cleavable moieties, allowing for a two-step amplification process to produce gapped, double-stranded amplicons, which are then repaired and amplified using universal primers, reducing primer dimers and enabling rapid production of highly multiplexed targeted libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional library preparation methods are used, then existing protocols can be followed, but the process is inefficient and does not allow for rapid production of highly multiplexed targeted libraries
Solution Approach 1:
The library preparation process is divided into distinct functional modules: adapter design with universal handles and target-specific sequences, selective amplification using target-specific primers, digestion to remove primers, repair of gapped amplicons, and final amplification with universal primers. This segmentation allows each step to be optimized independently and enables rapid multiplexed library production.
Solution Approach 2:
The invention employs universal handle sequences in the adapters that can be used across different target sequences, allowing the same universal primers to amplify multiple different targets. This universality reduces the need for multiple separate primer sets and enables high-throughput multiplexed library preparation from a single reaction.
2Measurement precision
If traditional amplification methods are used, then target sequences can be amplified, but primer dimers are generated that hinder accurate analysis
Solution Approach 1:
The invention extracts and removes the target-specific primer sequences from the final library through enzymatic digestion of the cleavable moieties. This extraction step eliminates primer dimers and unwanted primer sequences while retaining the target amplicons, thereby improving the accuracy of subsequent analysis.
Solution Approach 2:
The invention introduces cleavable moieties as intermediary elements that are temporarily incorporated into the amplicons during amplification but are subsequently removed through digestion. These intermediaries enable the amplification process while allowing for their later elimination to prevent primer dimer interference in final analysis.
3Adaptability or versatility
If multiple target sequences are amplified simultaneously, then multiplexing is achieved, but primer dimers and reaction complexity increase
Solution Approach 1:
The invention uses universal handle sequences and universal primers that can simultaneously amplify multiple different target sequences in a single reaction. This universality enables high multiplexing capability without proportionally increasing reaction complexity, as the same universal components work across all targets.
Solution Approach 2:
While using universal components, the invention incorporates target-specific sequences and cleavable moieties at specific locations in the adapters. This local differentiation allows each target to be specifically identified and amplified while maintaining the overall simplicity of using universal primers and handles across all targets.
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 and rapid production of highly multiplexed targeted libraries, suitable for various applications including sequencing, with reduced primer dimers and improved accuracy in detecting low-frequency alleles.
Implementation Method 1
a DNA polymerase to extend the primers to amplify the targets
Implementation Method 2
the amplicons are digested with a uracil DNA glycosylase
Implementation Method 3
the gapped amplicons are repaired with a DNA polymerase
Data Source
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AI summary
Provided are methods for preparing a library of target nucleic acid sequences, as well as compositions and uses therefor. Methods comprise contacting a nucleic acid sample with a plurality of adaptors capable of amplification of one or more target nucleic acid sequences under conditions wherein the target nucleic acid(s) undergo a first amplification; digesting the resulting first amplification products; repairing the digested target amplicons; and amplifying the repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the plurality of adaptor compositions comprise a handle and a targeted nucleic acid sequence and optionally one or more tag sequences. Provided methods may be carried out in a single, addition only workflow reaction, allowing for rapid production of highly multiplexed targeted libraries, optionally including unique tag sequences. Resulting library compositions are useful for a variety of applications, including sequencing applications.