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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Xiaoxing Lv1, Meiru Zhao1, Yuting Yi1
1Geneplus-Beijing Institute.
Abstract:
The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter's ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.
Insights
Enrich Rare Mutation Sequencing (ER-Seq) enhances the detection of rare mutations in circulating tumor DNA (ctDNA), improving sensitivity for clinical oncology applications.
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.
