Modified Primer Nucleic Acid Libraries for Adapter-Ligation Control
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Solution Overview
Problem
Current nucleic acid library preparation methods result in a significant fraction of non-fragmented DNA and primer dimers, leading to increased sequencing costs and reduced coverage, as they require more reads to compensate for insufficient sequencing of full DNA fragments.
Innovation Solution
A method using modified primers with blocking groups or nucleotide analogues to prevent adapter ligation and primer dimer formation, followed by endonuclease excision to generate a fragmented nucleic acid library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If fragmentation-based library preparation is used, then DNA fragments can be sequenced, but a significant fraction of non-fragmented DNA remains leading to uneven coverage
Solution Approach 1:
The method performs preliminary fragmentation of DNA molecules before library preparation, ensuring that DNA is pre-fragmented into appropriate sizes. This preliminary action prevents the formation of non-fragmented DNA in the final library, achieving uniform coverage without sacrificing sequencing reliability
Solution Approach 2:
The invention segments DNA molecules into smaller fragments through controlled fragmentation before adapter ligation. This segmentation ensures that all DNA molecules in the library are of appropriate size for sequencing, eliminating the uneven coverage problem caused by non-fragmented DNA while maintaining sufficient coverage depth
2Productivity
If adapter oligonucleotides are ligated to all polynucleotides, then library preparation is complete, but primer dimers and non-fragmented DNA are also sequenced increasing costs
Solution Approach 1:
The method applies different properties to different parts of the DNA population: fragmented DNA molecules receive adapter oligonucleotides and are sequenced, while non-fragmented molecules are blocked from adapter ligation or removed. This local differentiation ensures that only useful fragments are sequenced, improving sequencing efficiency while reducing waste of sequencing capacity on primer dimers and non-fragmented DNA
Solution Approach 2:
The invention extracts or removes non-fragmented DNA and primer dimers from the library before sequencing. By taking out these unwanted components, the method ensures that sequencing capacity is dedicated only to properly fragmented DNA molecules, reducing the number of reads needed and lowering sequencing costs while maintaining productivity
3Reliability
If the number of sequencing reads is increased to compensate for non-fragmented DNA, then coverage is improved, but costs increase
Solution Approach 1:
By performing preliminary fragmentation and selective adapter ligation, the method ensures that the final library contains only fragmented DNA molecules. This preliminary action eliminates the need to increase sequencing reads to compensate for non-fragmented DNA, achieving sufficient coverage at standard sequencing costs while maintaining high productivity
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
Reduces the number of non-fragmented nucleic acids and primer dimers, improving sequencing accuracy and reducing costs by allowing fewer sequencing reads to achieve full DNA coverage.
Implementation Method 1
the nucleotide analogue is excised after step d) by an endonuclease, thereby removing the primer binding site provided by the adapter oligonucleotides
Implementation Method 2
The functional group is either a blocking group at the 5′ end of said modified primer (FIG. 2), thereby preventing the ligation of the adapter oligonucleotides
Implementation Method 3
Amplification of the polynucleotides using a polymerase
Data Source
AI summary
The invention is directed to a method for obtaining a nucleic acid library of a sample comprising polynucleotides comprising the steps: a. Providing a plurality of modified primer to the polynucleotides, wherein said modified primer is a starting point for a polymerase for nucleic acid amplification b. Amplification of the polynucleotides using a polymerase c. Fragmentation of amplified polynucleotides, thereby obtaining a mixture of fragmented and un-fragmented polynucleotides comprising said modified primer d. Ligation of a plurality of adapter oligonucleotides to the mixture obtained in step c), thereby obtaining a mixture of polynucleotides comprising said adapter oligonucleotides, wherein said adapter oligonucleotides comprise a binding site for an amplification primer e. Providing an amplification primer to the mixture obtained in step d), wherein said amplification primer is a starting point for a polymerase for nucleic acid amplification f. initiate a nucleic acid amplification by providing a polymerase Characterized in that the modified primer provided in step a) comprises a functional group # wherein said functional group is a blocking group at the 5′ end of said modified primer, thereby preventing the ligation of the adapter oligonucleotides or # wherein said functional group is at least one nucleotide analogue, and wherein the nucleotide analogue is excised after step d) by an endonuclease, thereby removing the primer binding site provided by the adapter oligonucleotides, thereby preventing binding of the amplification primer provided in step f) and a nucleic acid amplification of fragmented polynucleotides comprising the modified primer