Title: Next-Generation Targeted Sequencing of Circulating Cell-Free DNA from Droplet Volumes of Blood 

Publication: International Journal of Life Sciences Research,Vol. 3, Issue 3, pp: (23-29) July–September 2015

Authors: Jonathan Spurgin, Andrew Ford, John Athan

Abstract: The non-invasive circulating cell-free DNA (cfDNA) approach – a liquid biopsy – is revolutionizing a paradigm shift in how cancer is detected, monitored and treated. In contrast to single-site, single time-point sampling by tissue biopsy, real-time and longitudinal mutation profile derived from tumor-specific cfDNA could potentially inform better and faster clinical decision-making, monitor tumor dynamics, assess response to treatment and identify mutations associated with acquired drug resistance. However, cfDNA analysis requires large volume of blood due to its relatively low amount in circulation and poor extraction efficiency of current methodologies. To overcome these major challenges, we have developed a proprietary cfDNA recovery technology with unique features of ultra-low input and ultra-high output. In this study, we evaluated our method side-by-side with the industry standard Qiagen kit, for the yield, cfDNA amplifiability and mutation detection from patient plasma.
Compared to Qiagen cfDNA extraction kit using different chemistry and different workflow, our approach allowed high-yield cfDNA enrichment directly from droplet volumes of unprocessed plasma, leading to >100-fold more recovery. NGS studies with cfDNA from 17 cancer plasma and 2 spiked samples further demonstrated the superiority of our protocol over Qiagen kit in generating more usable, on-target, high-quality ≥Q20 reads, and detecting more mutations.
Our cfDNA preparation breakthrough enables clinicians and laboratories to work with a sample volume as small as 20 microliters (via a finger-prick), in contrast to the current requirement of 10-20 milliliters, further expedite clinical decision-making and identify targeted therapies for eligible patients in a time- and cost-efficient manner.

Keywords: Cell-free DNA, liquid biopsy, blood-drop, next-generation sequencing.

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