Y-Shaped Nucleic Acid Adapters with UMIs for Sensitive ctDNA Detection
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Solution Overview
Problem
Existing methods for detecting and analyzing circulating tumor DNA (ctDNA) are limited by low DNA content in samples, leading to poor analytical sensitivity and high error rates, making it difficult to effectively monitor and profile cancer-related mutations.
Innovation Solution
The use of polynucleotide adapter compositions, specifically Y-shaped nucleic acid adapters with unique molecular identifiers, enhances ligation efficiency and allows for sensitive detection of ctDNA mutations by amplifying and sequencing low amounts of cell-free DNA, using biotin labeling and barcode sequences to track individual molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional library preparation methods are used, then the process is simple, but analytical sensitivity is poor and error rates are high due to low DNA content
Solution Approach 1:
The library preparation process is divided into separate strands (first and second strands) with unique molecular identifiers on each, allowing independent tracking and error correction. This segmentation enables better sensitivity by distinguishing true mutations from preparation errors.
Solution Approach 2:
Unique molecular identifiers serve as intermediary elements that link original DNA molecules to their amplified copies. These identifiers act as mediators to track individual molecules through the complex library preparation process, enabling error correction and improving measurement precision.
2Productivity
If standard adapter ligation is used, then the procedure is straightforward, but ligation efficiency is insufficient for low DNA content samples
Solution Approach 1:
The adapter design uses asymmetric structures with different arms (first and second strands) that have distinct unique molecular identifiers. This asymmetry optimizes ligation efficiency by preventing adapter dimer formation while maintaining ease of synthesis through standardized asymmetric adapter designs.
Solution Approach 2:
The adapter sequences are designed with specific parameter optimizations including unique molecular identifier lengths, spacer regions, and hybridization characteristics that enhance ligation efficiency for low input samples while remaining manufacturable.
3Reliability
If degenerate or semi-degenerate sequences are used in adapters, then sequence diversity is increased, but manufacturing precision and error rates worsen
Solution Approach 1:
Degenerate and semi-degenerate sequences are extracted and removed from the adapter design. By eliminating these error-prone elements, the patent achieves lower error rates and improved reliability while maintaining sufficient adaptability through the unique molecular identifier system.
Solution Approach 2:
Instead of using degenerate sequences to create diversity, the patent uses precise copying of known adapter sequences with unique molecular identifiers. This copying approach maintains manufacturing precision while providing the necessary diversity through the identifier sequences.
4Quantity of substance
If limited DNA input is used, then sample volume is reduced, but detection limits and analytical sensitivity deteriorate
Solution Approach 1:
Unique molecular identifiers are incorporated into adapters before ligation to DNA samples. This preliminary action enables subsequent error correction and sensitivity improvement without requiring additional DNA input, as the identifiers are already in place to track and validate molecules throughout the process.
Solution Approach 2:
The unique molecular identifiers provide feedback mechanisms that allow detection and correction of errors introduced during library preparation. This feedback system improves detection limits by distinguishing true low-abundance mutations from preparation artifacts, enabling sensitive detection even with limited DNA input.
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
The method improves detection limits and analytical sensitivity, enabling accurate profiling of ctDNA mutations in cancer-related genes even with limited DNA input, reducing sequencing errors and increasing the recovery of ctDNA from clinical samples.
Implementation Method 1
the first proximal region of the first oligonucleotide strand hybridizes with the second proximal region of the second oligonucleotide strand
Data Source
AI summary
The present technology provides polynucleotide compositions and methods of using the same to detect circulating tumor DNA (ctDNA) in a patient. Kits for use in practicing the methods are also provided.