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Abstract
Abstract Circulating cell free DNA has been shown to have the potential as a non-invasive substrate for the detection of cancer and its progression as well as the determination of therapeutic resistance. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing is an optimal tool for mutation detection. Here, we demonstrate a complete workflow from isolation through validation of circulating tumor DNA. We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation. We then completed molecular characterization of the isolated circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples complete with TaqMan® validation. Our results validate the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing and validation, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research. Citation Format: Dalia Dhingra, Xingwang Fang, Matt Carter, Dumitru Brinza, Charles Scafe, Christopher Davies, Fiona Hyland. A circulating cell free DNA workflow for targeted sequencing - from isolation through validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4931. doi:10.1158/1538-7445.AM2015-4931
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