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Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

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.

|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.

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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.