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11:11
Detection of Rare Mutations in CtDNA Using Next Generation Sequencing
Published on: August 24, 2017
Next-generation cDNA screening for oncogene and resistance phenotypes
Nobuaki Shindoh1, Akinori Yoda, Yuka Yoda
1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Plos One
|November 13, 2012
Summary
This study presents a new platform for functional screening of genetic alterations in cancer. It efficiently identifies DNA and RNA changes driving cancer growth and drug resistance using next-generation sequencing.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Next-generation sequencing (NGS) identifies numerous genetic alterations in cancer.
- Defining the functional impact of these alterations is crucial for targeted therapies.
- Existing methods may lack efficiency in screening small RNA samples.
Purpose of the Study:
- To develop and validate a novel platform for functional screening of genetic alterations.
- To identify DNA and RNA alterations conferring specific phenotypes like proliferation and drug resistance.
- To demonstrate the platform's broad applicability in cancer research.
Main Methods:
- Construction of full-length cDNA libraries from limited total RNA.
- Phenotypic screening of libraries in cytokine-dependent Ba/F3 cells for transformation and resistance.
- Deconvolution of screened libraries using barcoded next-generation sequencing.
Main Results:
- The platform efficiently identified DNA and RNA alterations conferring cytokine-independent proliferation and drug resistance.
- RNA alterations and intergenic fusions were successfully detected.
- Multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K, and an ERBB2 splice variant, were identified.
Conclusions:
- The developed cDNA library-based screening platform is effective for identifying functional genetic alterations.
- This approach enables simultaneous deconvolution of multiple libraries, accelerating discovery.
- The platform has broad potential for discovering phenotype-driving transcripts in vitro and in vivo.
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