Abstract 5391: Low frequency variant detection and tissue-of-origin exploration using liquid biopsies
Authors: Lenhart Justin S. , Wood Ashley , Sandhu Sukhinder , Schumacher Cassie , Kurihara Laurie , Makarov Vladimir , Harkins Tim 
Published: July 2017

Abstract
The promise of liquid biopsy assays lies in the non-invasive monitoring of diseases, such as cancer, through circulating, cell-free DNA (cfDNA) or circulating tumor cell DNA. This may assist in advancing early-stage diagnosis while simultaneously monitoring treatment response over time. Since these materials are often limited, most liquid biopsy assays incorporate targeted sequencing to enable cost-effective deep coverage of target loci for detection of low frequency pathogenic variants. Yet a critical aspect in attaining the necessary sensitivity is an assay that produces uniform, comprehensive coverage from low DNA input quantities. We have developed a liquid biopsy workflow to enable low frequency variant detection from a 10 mL blood draw using the Promega Maxwell RSC combined with Swift Biosciences Accel-NGS 2S® library preparation methodologies. In addition, we explore whether methylation patterns of the extracted cfDNA possess information of the tissue-of-origin.
Whole blood samples were collected in Streck cell-free DNA BCT vials from patients with late stage cancer and cfDNA was extracted with the Promega Maxwell RSC. This instrument yielded DNA outputs ranging from 8-32 ng, with a size profile defined by a predominant peak of ~170bp and a mean Alu repeat qPCR integrity score of 0.22, characteristic of high quality cfDNA lacking cellular DNA content. A total of 20 ng cfDNA was used to make an Accel-NGS 2S Hyb library followed by hybridization capture using Agilent SureSelect Human All Exon probes. The Accel-NGS 2S Hyb Kit exhibits a 90% library conversion rate with cfDNA and provides high complexity libraries with uniform target coverage. In addition, molecular barcodes were incorporated to label each library molecule uniquely prior to PCR amplification. These molecular barcodes were utilized for accurate removal of PCR duplicates while simultaneously preserving naturally occurring fragmentation and strand duplicates to maximize data recovery. Secondly, these barcoded molecules were grouped to generate consensus sequences after removal of false positives originating from PCR and sequencing errors. Variant calling was performed using Vardict and Lofreq enabling highly sensitive and precise detection of variants down to a 0.5% allele frequency.
In parallel, we have developed a workflow to determine if the epigenetic status of cfDNA can identify tissue-of-origin. This workflow utilizes the Accel-NGS Methyl-Seq DNA Library Kit to enable unbiased characterization from low (5 ng) cfDNA inputs. Through whole genome bisulfite sequencing, using a priori knowledge of differentially methylated regions characteristic of different human tissues, we can identify the predominant tissue source of cfDNA in blood.
Citation Format: Justin S. Lenhart, Ashley Wood, Sukhinder Sandhu, Cassie Schumacher, Laurie Kurihara, Vladimir Makarov, Tim Harkins. Low frequency variant detection and tissue-of-origin exploration using liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5391. doi:10.1158/1538-7445.AM2017-5391