Abstract 3637: Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies
Authors: Sandhu Sukhinder K. , Schumacher Cassie , Kurihara Laurie , Harkins Tim , Makarov Vladimir 
Published: July 2016

Abstract
Introduction
Hybridization-based target enrichment techniques coupled with Next Generation Sequencing (NGS) provide a useful and cost-efficient means to study disease specific target regions including whole exomes and gene panels. More than 500 whole exome analyses are behind The Cancer Genome Atlas (TCGA), which is the largest knowledge base for cancer studies including research, prevention, treatment, and care. Clinical samples may be limited in input and of compromised quality due to formalin fixation, however most current NGS library preparation methods require 50-100 ng high quality DNA for such studies. Here we present an efficient method which enables high quality target enrichment and variant calling from inputs as low as 1-25 ng.
Method
NGS libraries for hybridization capture were made from 1 to 100 ng of Coriell Hapmap samples (NA12878, etc), clinical Formalin Fixed Paraffin Embedded (FFPE) and Horizon Discovery (HDx) reference DNA (HDx 701) using the Accel-NGS® 2S Hyb DNA Library Kit. Amplified libraries were then enriched using specific targeted panels (xGen® Pan-Cancer and xGen® AML) or SeqCap™ EZ MedExome using manufacturer's specifications (IDT™ and Roche NimbleGen™). Enriched libraries were captured using streptavidin beads. Captured libraries were then amplified according to the manufacturer's specifications. Targeted panels were sequenced on an Illumina® MiSeq® using V2 chemistry and MedExome on a HiSeq® using V4 chemistry. Sequence analysis was performed with custom pipelines using BWA for alignment and GATK, Samtools, and Freebayes for variant calling.
Results
Sequencing analyses yielded a minimum average coverage of 30x and more than thirty fold enrichment. Enriched libraries exhibited significant sequence complexity with minimal duplicates and without any base-composition bias. The percent on-target varied with type of input and ranged from 50-80%. Germline variant calling results had &gt; 99% concordance with the NIST GIAB truth list with sensitivity and precision of &gt; 98%, even from inputs as low as 1 ng of DNA. Somatic variant calling down to 1-5% allele frequency was also evaluated at various DNA input quantities and greater depth of sequencing; results for FFPE and DNA standards will be presented.
Conclusions
Accel-NGS 2S Hyb technology can be used for whole exome and targeted enrichment studies from low quantity and low quality FFPE clinical samples. High complexity libraries with minimal bias yield high quality sequence data which enables discovery and detection of somatic variants in tumor samples to identify molecular drivers associated with different cancer types. The technique facilitates better understanding of complex cancer genomes and guide precision medicine.
Citation Format: Sukhinder K. Sandhu, Cassie Schumacher, Laurie Kurihara, Tim Harkins, Vladimir Makarov. Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3637.