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  • High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic-Based Multiplex PCR and Next-Generation Sequencing.

High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic-Based Multiplex PCR and Next-Generation Sequencing.

The Journal of molecular diagnostics : JMD (2017-09-05)
Yinghui Guan, Oleg Mayba, Thomas Sandmann, Shan Lu, Younjeong Choi, Walter C Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W R Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Mark R Lackner, Yulei Wang
ABSTRACT

Circulating tumor DNA (ctDNA) has potential to serve as a biomarker for noninvasive monitoring of treatment response and disease progression. However, broad clinical applicability of ctDNA has been limited by the low sensitivity, throughput, and patient coverage offered by existing ctDNA detection methods. Herein, we report the adaptation and characterization of the microfluidics multiplex PCR sequencing technology for high-throughput and sensitive quantitation of ctDNA. A multiplex PCR preamplification step was developed and incorporated into the microfluidics multiplex PCR sequencing work flow to enable low-input ctDNA analysis with enhanced sensitivity. An empirical bayesian model was developed to characterize both position and substitution-associated system errors specific to this platform and provided a tailored approach to greatly enhance the confidence and accuracy of variant calling for ctDNA analysis. Clinical validation of this platform for ctDNA mutation detection demonstrated an overall sensitivity of 92% and specificity of 100% when using mutation calls in the matched tumor tissues as a benchmark. Finally, we established an early proof of concept of clinical utility of this ctDNA work flow for monitoring disease progression using clinical trial samples. Our novel ctDNA work flow provides a high-throughput and sensitive platform that can be implemented in clinical trials for mutation detection and disease monitoring from plasma ctDNA.

MATERIALS
Product Number
Brand
Product Description

Roche
FastStart High Fidelity PCR System, 5 unit/μL, suitable for PCR, High Fidelity PCR, hotstart, Multiplex PCR