Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
| Autor: | Zhao Meiru, Xin Yi, Jianhui Ma, Rongrong Chen, Guan Yanfang, Ling Yang, Liu Tao, Yuting Yi, Lucheng Zhang, Lv Xiaoxing |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Genetics Clinical Oncology General Immunology and Microbiology General Chemical Engineering General Neuroscience DNA Mutational Analysis High-Throughput Nucleotide Sequencing Genetic data Biology General Biochemistry Genetics and Molecular Biology DNA sequencing Circulating Tumor DNA 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Single cell sequencing chemistry Mutation Rare mutations Humans Single mutation Exome sequencing DNA Cancer Biology |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/56342 |
| Popis: | 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 10(7) 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|