Loop-Shaped DNA Adapters for Sequencing Error Correction
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Solution Overview
Problem
Deep sequencing of circulating cell-free DNA for cancer detection is hindered by process-related errors, making it difficult to accurately identify rare variants indicative of cancer.
Innovation Solution
A method involving the use of loop-shaped double-stranded DNA adapters with unique molecular identifiers and endonuclease restriction sites to correct sequencing errors, generate consensus sequences, and identify rare variants by ligating adapters to DNA fragments, cleaving to produce single-strand molecules, and amplifying for sequencing libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If deep sequencing is performed to detect rare variants, then detection sensitivity is improved, but sequencing error rates increase making accurate identification difficult
Solution Approach 1:
The patent creates multiple copies of the original DNA fragment through PCR amplification after adapter ligation. Each copy carries the same sequencing information but allows independent error analysis. By comparing multiple copies (consensus building), the system can distinguish true variants from sequencing errors, thereby improving detection accuracy while maintaining the ability to identify rare variants at low frequencies.
Solution Approach 2:
The patent implements error correction through a feedback mechanism where sequence reads are grouped by UMI, and the consistency across multiple reads is evaluated. Reads that agree form a consensus sequence, while discordant reads (likely errors) are resolved through majority voting or manual review. This feedback loop continuously refines the accuracy of variant identification.
2Reliability
If unique molecular identifiers are added to adapters, then error correction capability is improved, but library preparation complexity increases
Solution Approach 1:
The patent merges multiple functions into the adapter structure: the adapter serves as both a sequencing primer binding site and a carrier for the UMI tag. This integration allows error correction capability to be added without requiring separate components for each function, thereby limiting the increase in overall system complexity while achieving the desired error correction.
Solution Approach 2:
The patent changes the parameter of the adapter from a simple sequencing primer to a complex structure containing UMI and restriction sites. This parameter change enables error correction and sample tracking capabilities. The additional complexity is localized to the adapter rather than propagating through the entire library preparation workflow.
3Measurement precision
If loop-shaped adapters with restriction sites are used, then sequencing accuracy is improved, but manufacturing complexity increases
Solution Approach 1:
The adapter is segmented into distinct functional regions: the UMI segment for error correction, the restriction site segment for controlled cleavage, and the sequencing primer binding segment. This segmentation allows each region to be optimized independently and simplifies the manufacturing process by enabling modular synthesis and quality control of individual components before final assembly.
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 enhances the accuracy of identifying rare variants by correcting sequencing errors and improving the detection of cancer-related genetic alterations, enabling effective cancer diagnosis and monitoring.
Implementation Method 1
The DNA polymerase may be a Klenow fragment, a Taq polymerase, a Pfu polymerase, or any other DNA polymerase known in the art
Implementation Method 2
The first loop-shaped adapter may be cleaved with an endonuclease to generate a plurality of linear single-strand DNA (ssDNA) molecules
Data Source
AI summary
Aspects of the invention include methods for preparing sequencing libraries, performing sequencing procedures that can correct for process-related errors, and identifying rare variants that are or may be indicative of cancer.