Nucleic Acid Library Enrichment via Strand Neutralization
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Solution Overview
Problem
Current genome sequencing methods face inefficiencies due to high rates of artefactual antisense reads, which increase sequencing costs and reduce the fraction of bona fide reads, particularly in high-throughput sequencing technologies.
Innovation Solution
A method is developed to enrich nucleic acid sequencing libraries by transforming double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA), neutralizing unwanted strands, and then capturing and reamplifying the desired ssDNA fragments to produce an enriched dsDNA library, thereby increasing sequencing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hybrid-capture technology is used to pull-down library molecules of interest, then the ability to enrich targeted regions is improved, but artefactual antisense reads are generated at a high rate
Solution Approach 1:
The library molecules are segmented into sense and antisense strands, with only the desired antisense strand being captured. The sense strand is neutralized and discarded, separating the useful component from the harmful artefactual reads.
Solution Approach 2:
The harmful sense strand is extracted and neutralized from the library before capture, preventing it from generating artefactual antisense reads during sequencing while retaining the useful antisense strand for enrichment.
2Measurement precision
If capture oligonucleotides are used to enrich target fragments, then the fraction of target fragments producing bona fide reads is increased, but sequencing costs increase due to discarded wasted reads
Solution Approach 1:
The sense strand is neutralized in advance before the capture step, preventing it from interfering with sequencing. This preliminary action ensures that all captured fragments will produce bona fide reads, eliminating wasted sequencing reads and reducing costs.
3Stability of the object's composition
If dsDNA libraries are used for sequencing, then the library structure is stable, but library molecules change orientation causing artefactual antisense reads
Solution Approach 1:
The problematic sense strand is extracted and neutralized from the dsDNA library, eliminating the source of artefactual reads while maintaining the stable dsDNA structure of the desired antisense strand for sequencing.
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 significantly increases the fraction of bona fide reads while reducing artefactual antisense reads, leading to improved sequencing efficiency and cost-effectiveness by enhancing the percentage of reads mapped confidently to the targeted transcriptome.
Implementation Method 1
the capture oligonucleotide hybridizes to the second strand of the non-artefactual dsDNA library member
Implementation Method 2
the capture oligonucleotide comprises a bait molecule having binding affinity to a binding partner, wherein the capture oligonucleotide hybridizes to the second strand
Data Source
AI summary
Provided herein are methods for enrichment of nucleic acid libraries for non-artefactual on-target molecules that produce bona fide sequencing reads while eliminating or reducing artefactual on-target molecules that produce wasted reads thereby improving sequencing efficiency. Methods comprise the neutralization of a single artefactual strand of a dsDNA molecule and capture of the non-artefactual strand thereby generating enriched sequencing libraries.