Abstract

A Complete Next-Generation Sequencing Workflow for Circulating DNA Isolation and Analysis

Xingwang Fang, Senior Staff Scientist, Thermo Fisher Scientific

Circulating cell-free DNA (cfDNA) has been shown to have potential as a non-invasive substrate for the detection and progression of tumor cells. As circulating tumor DNA is often present at low frequencies within cfDNA, targeted sequencing is an optimal tool for mutation detection.  To support advancement of cfDNA research, we have developed MagMAXTM Cell-free DNA Isolation kit to efficiently isolate and enrich cfDNA and Ion AmpliSeqTM technology to analyze rare mutation with deep sequencing.

We demonstrate a complete workflow providing:  1) isolation of cfDNA from plasma using the Applied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit either manually with a magnetic stand or automated on the Thermo Scientific™ KingFisher™ Flex or KingFisher™ Duo Prime instrument, followed by 2) molecular characterization of isolated cfDNA using the multiplexing capabilities of Ion AmpliSeq™ technology and the Ion PGM™ System.

cfDNA isolated with the MagMAX cfDNA Kit, the Ion AmpliSeq™ Cancer Hotspot Panel v2 demonstrates reproducible amplicon representation and variant detection across 50 genes of interest, covering 2,800 COSMIC mutations. The performance is comparable whether cfDNA is isolated with the MagMAX cfDNA Kit or a column-based protocol. Importantly, performance is also comparable when using less than half the volume of plasma with the MagMAX cfDNA Kit than with column-based protocols. Through saturation studies and subsampling, the limit of detection of hotspots in cfDNA on the Ion PGM System is determined to be below 1%.

This robust workflow for cfDNA isolation through targeted sequencing combines simple sample preparation with the ease of Ion AmpliSeq technology and the rapid turnaround of the Ion PGM System.