Blocker Oligonucleotide Hybridization for Adapter-Dimer Inhibition
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Solution Overview
Problem
Adapter-dimers in sequencing libraries negatively affect sequencing quality by being amplified and retained during enrichment steps, leading to inefficient use of reagents and time.
Innovation Solution
A method involving the use of blocker oligonucleotides that hybridize with excess unligated adapters, followed by ligation to form a hybridized complex, reducing the number of unligated adapters and inhibiting their amplification through additional steps such as enzymatic cleavage or gel purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If adapter ligation is performed to attach adapters to insert sequences, then sequencing library production is enabled, but adapter-dimers are formed as unwanted by-products
Solution Approach 1:
The patent introduces an intermediary oligonucleotide that acts as a blocking agent during adapter ligation. This intermediary binds to excess adapters, preventing them from forming dimers while allowing the desired adapter-insert ligation to proceed. The intermediary serves as a mediator that resolves the conflict between enabling library production and preventing harmful dimer formation.
Solution Approach 2:
The patent applies preliminary anti-action by adding blocking oligonucleotides before adapter-dimer formation can occur. These blockers preemptively bind to excess adapters, preventing the harmful ligation reaction between adapters. By acting in advance, the blockers prevent the formation of adapter-dimers before they can interfere with sequencing quality.
2Quantity of substance
If amplification reactions are used to enrich insert sequences, then target enrichment is achieved, but adapter-dimers are also amplified
Solution Approach 1:
The patent extracts or removes the harmful element (adapter-dimers) from the system before amplification. By using blocking oligonucleotides to bind and sequester excess adapters, the method prevents adapter-dimer formation in the first place. This extraction approach ensures that only desired insert sequences remain available for amplification, eliminating the problem of co-amplification.
Solution Approach 2:
The patent converts the potentially harmful excess adapters into a beneficial state by having them bind to blocking oligonucleotides. Instead of allowing these excess adapters to form dimers and be amplified, they are redirected to bind with blockers, which prevents their harmful effects while maintaining the enrichment of target sequences. The harm is transformed into a controlled interaction that benefits the overall sequencing process.
3Manufacturing precision
If hybridization capture is used for target enrichment, then non-target sequences are reduced, but adapter-dimers are retained and reagents are wasted
Solution Approach 1:
The patent performs preliminary action by preventing adapter-dimer formation before the hybridization capture step. Blocking oligonucleotides are added during the ligation phase to prevent dimer formation, so that when hybridization capture occurs, there are fewer adapter-dimers to be retained. This preliminary prevention reduces reagent waste during subsequent enrichment steps.
Solution Approach 2:
The patent extracts adapter-dimers from the mixture through blocking, preventing them from participating in hybridization capture. By removing or sequestering excess adapters with blocking oligonucleotides, the method ensures that hybridization capture focuses only on target sequences, eliminating the waste of reagents on capturing non-target adapter-dimers.
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
Significantly reduces the formation and amplification of adapter-dimers, improving sequencing library purity and efficiency by enhancing the incorporation of target nucleic acids into sequencing libraries.
Implementation Method 1
combining sample nucleic acids, each having a 5' end and a 3' end, with 3' adapters each having a 5' end portion at least 8 nucleotides in length and a 5' terminus which is an adenylated nucleotide, and a ligase, in a mixture under ligation reaction conditions, producing 1) first ligation products each including the 3' end of a sample nucleic acid ligated to the 5' terminus of a 3' adapter
Implementation Method 2
incubating the mixture under hybridization conditions, thereby hybridizing the first blocker oligonucleotide 3' portion with the second blocker oligonucleotide 3' portion, hybridizing the first blocker oligonucleotide 5' portion with the excess unligated 3' adapters, and thereby hybridizing the second blocker oligonucleotide 5' portion with the excess unligated 3' adapters
Data Source
AI summary
Methods of reducing adapter-dimers in a sequencing library are provided according to aspects of the present disclosure, including: hybridizing excess 3′ adapters to blocker oligonucleotides forming a hybridized complex, such that the excess 3′ adapters are unavailable to form adapter-dimers, the hybridized complex including: a first blocker oligonucleotide 3′ terminus adjacent to an adenylated nucleotide at 5′ terminus of a first unligated 3′ adapter and a second blocker oligonucleotide 3′ terminus adjacent to an adenylated nucleotide at 5′ terminus of a second unligated 3′ adapter, wherein the first blocker oligonucleotide has a 5′ portion complementary to and hybridized to the first unligated 3′ adapter, the second blocker oligonucleotide has a 5′ portion complementary to and hybridized to the second unligated 3′ adapter, and the first blocker oligonucleotide has a 3′ portion complementary to, and hybridized to, a 3′ portion of the second blocker oligonucleotide.