Multiplex Target Library Preparation by Blocking Abundant Sequences
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Solution Overview
Problem
Existing methods for detecting low-frequency target sequences in multiplex reactions are hindered by abundant sequences, requiring complex workflows and specialized reagents, which reduce the effectiveness of analyzing rare sequences.
Innovation Solution
A method involving a single amplification reaction with target-specific primers, including cleavable groups, followed by cleavage and adapter ligation, or a two-step amplification process with adapter-amplification, to prepare a library of target sequences, minimizing interference from abundant sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If complex workflows and specialized reagents are used to detect low-frequency target sequences, then detection capability is improved, but workflow complexity and cost increase
Solution Approach 1:
The patent divides the detection workflow into distinct functional modules: (1) multiplex amplification of target sequences, (2) selective digestion of abundant sequences using sequence-specific nucleases, (3) adapter ligation, and (4) sequencing. This segmentation allows each module to be optimized independently and simplifies the overall workflow by eliminating the need for complex specialized reagents while maintaining high detection precision for low-frequency targets
Solution Approach 2:
The patent extracts and removes abundant background sequences from the sample through selective nuclease digestion before library preparation. By taking out these interfering abundant sequences, the method enhances the relative proportion of low-frequency target sequences, improving detection capability without requiring complex specialized reagents or workflows
2Measurement precision
If complex workflows and specialized reagents are used to detect low-frequency target sequences, then detection capability is improved, but reagent cost increases
Solution Approach 1:
The patent employs universal reagents that can process multiple different target sequences simultaneously. The adapter sequences, nuclease enzymes, and amplification conditions are designed to be universal across different target types, eliminating the need for expensive specialized reagents for each specific target while maintaining high detection capability for low-frequency sequences
Solution Approach 2:
The patent changes the approach from using specialized high-cost reagents to using standard, cost-effective reagents in combination with selective digestion. By modifying the workflow parameters to include sequence-specific nuclease treatment, the method achieves high detection precision using inexpensive, widely available reagents
3Productivity
If abundant sequences are present in the sample, then library complexity is increased, but detection of rare sequences is reduced
Solution Approach 1:
The patent converts the harmful effect of abundant sequences into a benefit by using their known sequence characteristics as targets for selective nuclease digestion. The abundance of these sequences provides enough target sites for the nucleases to efficiently cleave them, thereby removing the interference and enhancing the detection of rare sequences while maintaining library complexity
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
Enables efficient production of nucleic acid libraries for analyzing rare sequences by simplifying the workflow and reducing interference from high-abundance sequences, enhancing the detection of low-frequency alleles.
Implementation Method 1
amplifying within a single amplification reaction mixture a multiplex of different target sequences from a sample including a plurality of different target sequences, wherein the amplifying includes contacting at least a portion of the sample with a plurality of target-specific primers, and a polymerase under amplification conditions
Implementation Method 2
cleaving the cleavable groups of the multiplex of different amplified target sequences and forming cleaved ends
Implementation Method 3
ligating at least two adapters to cleaved ends of at least one of the multiplex of different amplified target sequences
Implementation Method 4
none of the adapters in the ligation reaction hybridizes under high stringency conditions to any one of the multiplex of different amplified target sequences
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
Efficient methods for production of targeted libraries from complex samples is desirable for a variety of nucleic acid analyses. Provided are methods of selectively blocking abundant targets present in a sample for preparing libraries of target nucleic acid sequences, thereby allowing for rapid production of highly multiplexed targeted libraries and analysis of low frequency sequences, including sequencing applications. Methods optionally include use of unique tag sequences. Methods comprise contacting a nucleic acid sample with a plurality of target specific primers or adapters 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; ligating the digested target amplicons or repairing the digested target amplicons; and amplifying the ligated or repaired products in a second amplification, thereby producing a library of target nucleic acid sequence. Each of the reactions further comprise target specific primers that are not capable of completely processing the workflow, resulting in non-useful amplicon production and thereby blocking selected target sequences, e.g., those present in high abundance in the sample. 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, which are optimized for detection of low frequency target sequences.