Nucleic Acid Library Normalization via Hybridization Capture
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Solution Overview
Problem
Current methods for nucleic acid library normalization struggle with efficiently reducing the concentration of high abundance species, leading to redundant sequencing efforts and inefficiencies in assessing diverse nucleic acid samples.
Innovation Solution
The method involves hybridizing oligonucleotides with a binding moiety to nucleic acid molecules, generating complementary strands, denaturing, partially reannealing, and using a capture molecule on a solid support to remove reannealed strands, thereby reducing the abundance of high concentration species, allowing for a more balanced representation of low abundance species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional sequencing methods are used on unnormalized libraries, then high abundance species dominate the sequencing reads, but low abundance species are underrepresented requiring redundant sequencing efforts
Solution Approach 1:
The patent extracts high abundance species from the nucleic acid library by hybridizing them to complementary strands with capture moieties, then removing these hybridized complexes via capture molecules on solid supports. This extraction of dominant species allows low abundance species to be enriched and properly represented in sequencing without requiring redundant efforts.
Solution Approach 2:
The patent introduces asymmetry by differentially labeling the two strands of double-stranded nucleic acids - one strand receives a capture moiety while the other remains unlabeled. This asymmetric labeling enables selective capture and removal of specific strands, thereby normalizing the library composition to balance representation across all species.
2Reliability
If library normalization is not performed, then the entire catalogue of nucleic acids cannot be fully assessed, but normalization requires additional processing steps
Solution Approach 1:
The patent performs preliminary normalization processing before sequencing by pre-hybridizing, extending, and capturing strands in advance. This preliminary action ensures that when sequencing occurs, the library is already normalized and ready for efficient assessment of the entire nucleic acid catalogue without requiring complex real-time adjustments during sequencing.
Solution Approach 2:
The patent introduces intermediary molecules including oligonucleotides with capture moieties,延伸 enzymes, and solid support capture molecules. These intermediaries facilitate the normalization process by mediating the selective binding and removal of high abundance species, making the overall process more manageable despite the multiple steps involved.
3Loss of energy
If high abundance species are not reduced, then sequencing resources are wasted on redundant reads, but reducing them requires hybridization and capture steps
Solution Approach 1:
The patent applies partial action by performing hybridization and capture steps only to the extent necessary to reduce high abundance species to appropriate levels. The process is optimized to remove sufficient dominant species without over-processing, thereby conserving sequencing resources while avoiding unnecessary complexity in the normalization procedure.
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 normalizes nucleic acid libraries by significantly reducing the content of high abundance species, enhancing the representation of low abundance species, and optimizing sequencing efforts by ensuring more balanced sequencing reads.
Implementation Method 1
hybridizing a plurality of first oligonucleotides comprising a binding moiety with a first plurality of nucleic acid molecules
Implementation Method 2
extending the plurality of first oligonucleotides to generate a plurality of complementary strands of the first plurality of nucleic acid molecules
Implementation Method 3
denaturing a plurality of double-stranded nucleic acid molecules comprising the plurality of complementary strands of the first plurality of nucleic acid molecules
Implementation Method 4
partially reannealing the plurality of complementary strands of the first plurality of nucleic acid molecules
Implementation Method 5
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 binding moiety
Data Source
AI summary
The disclosure provides for methods, compositions, and kits for normalizing nucleic acid libraries, for example sequencing libraries.