Nucleic Acid Library Preparation with 3′ Adaptors and Strand UIDs
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for sequencing, particularly in genetically heterogeneous mixtures such as tumors or mixed microbial populations, suffer from high error rates due to PCR or sequencing errors, and are inefficient for targeted amplicon sequencing, especially for small fragments like plasma DNA, small RNA, or miRNA, leading to false negatives and positives.
Innovation Solution
The method involves adding an adaptor sequence to the 3' end of single-stranded nucleic acids using an adaptor template oligonucleotide (ATO) with a 3' random sequence and a universal sequence, allowing for accurate extension and sequencing by polymerase, followed by removal of the ATO to reduce errors, and using unique identifiers (UIDs) to tag each strand independently for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiplex PCR is used for targeted sequencing of large complex fragments, then sequencing coverage is improved, but false positive and false negative results increase due to non-specific priming and amplification artifacts
Solution Approach 1:
The patent divides the sequencing process into two independent strands, sequencing each strand separately and independently. This segmentation allows errors in one strand to be identified and corrected by comparing with the other strand, thereby reducing false positives and false negatives while maintaining sequencing coverage.
Solution Approach 2:
The patent implements a feedback mechanism where sequencing results from one strand are used to verify and correct results from the other strand. By requiring consensus between both strands for mutation calling, the system feedback-reduces amplification artifacts and non-specific priming errors.
2Ease of manufacture
If traditional adaptor ligation is used for targeted amplicon sequencing, then library preparation is achieved, but sequencing information in primer binding regions is lost and fusion/translocation events cannot be detected
Solution Approach 1:
Instead of ligating adaptors to the 5' end of amplicons (traditional approach), the patent adds adaptors to the 3' end of the amplicons. This inversion of the adaptor addition strategy preserves the primer binding regions for sequencing while still enabling library preparation and NGS compatibility.
Solution Approach 2:
The patent designs universal adaptor sequences that can be added to all amplicons regardless of their specific target region. These universal adaptors enable subsequent PCR amplification and sequencing while preserving the ability to detect fusion and translocation events through the retained primer binding regions.
3Measurement precision
If deep sequencing is performed on genetically heterogeneous mixtures, then mutation detection sensitivity is improved, but sequencing errors increase due to the ~1% error rate
Solution Approach 1:
The patent segments the DNA into two separate strands and sequences each independently with unique identifiers. By analyzing mutations in both strands, the system can distinguish true mutations (present in both strands) from sequencing errors (present in only one strand), thereby maintaining high sensitivity while reducing error rates.
Solution Approach 2:
The patent creates a copy of each strand with a unique identifier and sequences both copies. This copying approach allows for error correction by comparing the original strand sequence with its complementary strand sequence, filtering out sequencing errors while preserving true mutations.
4Measurement precision
If primers are designed for small target fragments like plasma DNA or miRNA, then targeted sequencing is achieved, but primer design freedom is limited and some regions become un-sequencable
Solution Approach 1:
The patent inverts the traditional approach by adding adaptors to the 3' end rather than relying on primers for adaptor incorporation. This allows primers to be designed with maximum flexibility for small targets without worrying about adaptor interference, while still enabling NGS library preparation through the 3' adaptor addition.
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 reduces sequencing errors by independently sequencing each strand, enabling accurate detection of rare mutations and mutations in heterogeneous samples, and allows for precise determination of nucleic acid copy numbers.
Implementation Method 1
The oligonucleotide is incubated with a population of target polynucleotides under conditions allowing hybridization of the 3' end of the target polynucleotides to the adaptor template oligonucleotide
Implementation Method 2
The mixture is incubated under conditions allowing extension of the hybridized 3' end of the target polynucleotides
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
This invention relates to methods, compositions and kits for extending a polynucleotide and for preparing sequencing library of polynucleotides involving generating modified target polynucleotide on an adaptor template oligonucleotide and tagging one or two strands of a target sequence. The sequencing library is suitable for massive parallel sequencing and comprises a plurality of double-stranded nucleic acid molecules.