Detection of Rare Mutations in CtDNA Using Next Generation Sequencing.

Area of Science:

• Oncology
• Genetics
• Molecular Biology

Background:

• Circulating tumor DNA (ctDNA) analysis via next-generation sequencing (NGS) is crucial for clinical oncology.
• Low sensitivity and potential for false results limit current ctDNA analysis.
• Detecting trace amounts of ctDNA is challenging but vital for understanding disease heterogeneity.

Purpose of the Study:

• To introduce Enrich Rare Mutation Sequencing (ER-Seq) for improved ctDNA analysis.
• To enhance the sensitivity and reliability of detecting rare mutations in ctDNA.
• To provide a cost-effective and universal method for accumulating genetic data.

Main Methods:

• ER-Seq employs unique sequencing adapters for efficient ctDNA recovery and bidirectional error correction.
• A selection of 1021 kb probes enriches target regions covering over 95% of tumor-related driver mutations.
• The method includes detailed protocols for probe design, library construction, target DNA capture, and data analysis.

Main Results:

• ER-Seq can distinguish a single mutation among 1 x 10^7 wild-type nucleotides.
• The technique efficiently filters background errors for precise rare mutation detection.
• A case study demonstrates the comprehensive workflow and successful data accumulation.

Conclusions:

• ER-Seq significantly improves the feasibility and reliability of ctDNA detection.
• This method is a promising tool for studying disease heterogeneity and clinical applications.
• The ER-Seq protocol is cost-effective, universal, and can be completed in 1-2 days.