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Updated: May 17, 2026
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.
Abstract:
There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.
Insights
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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