NGS Library Construction for Low-Frequency Mutation Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting low-frequency DNA mutations in next-generation sequencing face challenges such as high false positive rates and inefficient target enrichment, leading to wastage of sequencing data due to the low concentration of mutant DNA in samples.
Innovation Solution
A method involving end-repairing, A-tailing, and adapter addition to DNA fragments, followed by single-round PCR amplification using specific primers, to construct a next-generation sequencing DNA library, which reduces false positives and enhances enrichment efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR is used for target enrichment, then amplification can be achieved, but target enrichment efficiency is low and sequencing data is wasted
Solution Approach 1:
The patent performs end-repair and A-tailing of DNA fragments before adapter ligation, creating optimal conditions for subsequent PCR amplification. This preliminary preparation of DNA ends enables more efficient target enrichment in the single-round PCR step, directly addressing the low enrichment efficiency problem while reducing sequencing data waste.
2Productivity
If multiple PCR amplification rounds are conducted, then amplification strength is increased, but false positive rate increases
Solution Approach 1:
The patent employs a single-round PCR amplification strategy instead of multiple rounds, using optimized primer concentrations and extension conditions to achieve sufficient amplification in one step. This partial action approach maintains amplification strength while minimizing the accumulation of amplification errors and false positives that would occur with multiple rounds.
3Reliability
If single-round PCR amplification is used, then false positives are reduced, but amplification coverage may be insufficient
Solution Approach 1:
The patent optimizes multiple parameters including PCR extension time (15-30 seconds), primer concentrations (0.2-1.0 μM), and polymerase activity to ensure sufficient amplification coverage is achieved within a single round. These parameter adjustments compensate for the reduced number of amplification cycles while maintaining high reliability.
4Ease of manufacture
If standard adapter ligation is used, then library construction can proceed, but target enrichment efficiency remains low
Solution Approach 1:
The patent performs A-tailing of DNA fragments before adapter ligation, creating protruding 3' A-ends that are complementary to the 5' T-ends of adapters. This preliminary modification enables more efficient and specific adapter ligation, significantly improving target enrichment efficiency while maintaining library construction feasibility.
Data Source
AI summary
The present invention provides a method for constructing a next-generation sequencing library for detecting low-frequency mutations, and a kit thereof. The constructing method comprises steps of obtaining blunt-end DNA fragments, obtaining DNA fragments with A-tail at the 3′ end, obtaining adapter-added DNA fragments using a specific nucleotide sequence and obtaining amplification products using a specific nucleotide sequence.