Multi-position Double-tag Adapter Set for Gene Mutation Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current sequencing methods struggle to accurately detect somatic mutations in tumor cells due to high error rates and low signal-to-noise ratios, making it difficult to distinguish between noise and actual mutation sites.
Innovation Solution
A multi-position double-tag adapter set is introduced, which uses two different unique molecular identifiers (UMIs) on DNA strands to correct sequencing errors and improve mutation detection sensitivity, reducing base error rates and enhancing the signal-to-noise ratio for accurate detection of low-frequency mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed with standard procedures, but the base error rate remains high (1/1000-1/100) and somatic mutations cannot be accurately detected
Solution Approach 1:
The adapter is divided into multiple functional segments: a double-tag region with two different UMIs (first and second unique molecular identifiers), a restriction site, and a position tag. This segmentation allows each component to perform its specific function - the dual UMI system corrects sequencing errors while the position tag maintains proper orientation and diversity
Solution Approach 2:
The patent introduces an intermediary molecular marker (the multi-position double-tag adapter) that bridges the sample DNA and the sequencing process. The adapter's dual UMI system acts as an intermediary that tracks and corrects errors introduced during library preparation and sequencing, enabling accurate detection of low-frequency somatic mutations
2Measurement precision
If traditional bioinformatics methods are used, then data analysis is simpler, but the signal-to-noise ratio is too low to distinguish tumor mutations from systematic errors
Solution Approach 1:
The patent adds another dimension to the traditional single UMI approach by implementing a dual UMI system on opposite strands of the adapter. This dimensional expansion creates a more robust error correction mechanism where mutations must occur independently in both UMIs to be considered errors, significantly improving the signal-to-noise ratio for somatic mutation detection
Solution Approach 2:
The adapter is designed with pre-positioned UMIs and position tags that are incorporated during library preparation before sequencing. This preliminary action ensures that each DNA molecule is tagged with unique identifiers that track it through amplification and sequencing, enabling post-sequencing error correction without adding complexity to the sequencing process itself
3Reliability
If PCR amplification is performed during library construction, then sufficient DNA quantity is obtained, but mutations are introduced by PCR enzymes
Solution Approach 1:
The patent applies beforehand cushioning by incorporating a dual UMI system into the adapter before PCR amplification. This allows the system to pre-track each DNA molecule and subsequently correct PCR-introduced errors during data analysis. The position tag with restriction site also cushions against amplification biases by ensuring proper fragment orientation and size selection
Data Source
AI summary
A multi-position double-tag adapter set for detecting gene mutation and preparation method therefor and application thereof, the multi-position double-tag adapter set comprising a double-tag adapter A, a double-tag adapter B and a double-tag adapter C. The double-tag adapter A, the double-tag adapter B and the double-tag adapter C are obtained respectively by hybridizing an adapter primer P5 with an adapter primer P7-A, an adapter primer P7-B and an adapter primer P7-C 5′ ends of which are all modified with biotin. Using the multi-position double-tag adapter set, the mutation rate of 1×10−5 genes may be accurately detected and the sensitivity of gene mutation detection may be effectively improved. A plurality of mutation sites of a plurality of genes may be detected by one-time sequencing in combination with throughput of high-throughput sequencing.