Nucleic Acid Library Normalization via Affinity Capture
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Solution Overview
Problem
Current methods for assessing gene expression profiles through next-generation sequencing (NGS) face challenges due to PCR amplification biases, where high abundance species dominate, skewing the native gene expression profile and increasing sequencing costs.
Innovation Solution
The method involves hybridizing oligonucleotides with an affinity moiety to nucleic acid molecules, generating complementary strands, denaturing, and partially reannealing to remove high abundance species using capture molecules immobilized on a solid support, thereby generating a normalized nucleic acid library with reduced high abundance species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is performed in excess cycles to adequately amplify low expressers, then adequate sample amount is obtained, but the native gene expression profile is skewed by dominating high expresser PCR products
Solution Approach 1:
The patent divides the nucleic acid population into different abundance categories (high, intermediate, low expressers) and applies differential handling strategies. By segmenting the sample based on abundance and applying selective amplification or depletion strategies to different segments, the method preserves the true expression profile while ensuring adequate representation of all gene categories.
Solution Approach 2:
The patent extracts or removes high abundance species (high expresser genes) from the nucleic acid library through selective depletion. This extraction process reduces the skewing effect of highly abundant sequences on sequencing results, allowing low and intermediate expressers to be properly represented in the final analysis.
2Measurement precision
If Molecular Indexing is used to correct for PCR bias, then gene expression accuracy is improved, but sequencing cost increases due to high expressers dominating the sequencing run
Solution Approach 1:
The patent extracts high abundance species from the nucleic acid library before sequencing. By removing these dominant sequences that would otherwise consume the majority of sequencing reads, the method enables more efficient use of sequencing capacity to detect low and intermediate expressers, reducing the cost per useful measurement.
Solution Approach 2:
The patent performs preliminary depletion of high abundance species before the sequencing step. This preliminary action prepares the sample in advance, ensuring that the sequencing run is optimized for detecting lower abundance transcripts from the outset, thereby reducing the total sequencing cost required to achieve adequate coverage.
3Quantity of substance
If high abundance species are removed from the nucleic acid library, then sequencing cost-effectiveness is improved, but the representation of low abundance species must be maintained
Solution Approach 1:
The patent applies different qualities or treatments to different parts of the nucleic acid population. High abundance species receive selective depletion treatment, while low and intermediate expressers receive different handling (such as selective amplification or protected representation). This localized differentiation ensures cost-effectiveness through high abundance removal while preserving low abundance representation.
Solution Approach 2:
The patent applies partial depletion of high abundance species rather than complete removal, and uses selective amplification strategies for low abundance species. By applying partial action (depletion only to the extent necessary to reduce skewing) and excessive action (amplification beyond what would be needed for simple representation), the method achieves both cost-effectiveness and adequate low abundance representation.
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 effectively reduces the representation of high abundance species to less than 50% in the nucleic acid library, increasing the sequencing reads for low abundance species and improving the cost-effectiveness of sequencing by decreasing the dominance of high expresser genes.
Implementation Method 1
hybridizing a plurality of first oligonucleotides comprising an affinity moiety with a first plurality of nucleic acid molecules
Implementation Method 2
denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands
Implementation Method 3
partially reannealing the plurality of complementary strands of the first plurality of nucleic acid molecules
Implementation Method 4
removing the reannealed complementary strands of the first plurality of nucleic acid molecules by a capture molecule immobilized on one or more solid support to generate a second plurality of nucleic acid molecules, wherein the capture molecules specifically bind to the affinity moiety
Data Source
AI summary
This disclosure provides methods and compositions for removing one or more high abundance species from a plurality of nucleic acid molecules. In some embodiments, the methods and compositions can be used for normalizing nucleic acid libraries. In some embodiments, molecular labels are used in conjunction with the methods and compositions disclosed herein to improve sequencing efficiency.