Comparative evaluation of Agilent’s SureSelect platforms for FFPE
Generation of high-quality data from low input FFPE
Poster from AGBT 2018
Whole exome sequencing (WES) remains a cost effective way to capture regions of the genome that encode frequent disease-causing mutations. While intact DNA performs well, DNA from formalin-fixed, paraffin- embedded (FFPE) tissues presents certain challenges, with variable sample quality and limited yields often preventing successful library preparation from highly degraded samples. As most standard commercially-available kits require large DNA input amounts (≥200ng), target enrichment methods capable of handling low quality and/or low input samples while still generating high quality data are needed. Sample input limitations can be overcome by increasing the number of PCR cycles, at a cost of higher degrees of PCR duplication and reduction of library complexity, which compromise the template sequence representation. To limit the impact of PCR duplication on downstream analysis, we evaluated the use of molecular barcodes in the Agilent SureSelectXT HS target enrichment system to remove amplification artifacts, enabling detection of lower frequency somatic mutations. We compared library read breakdown data from our current WES protocols on matched FFPE and intact DNA samples to improve FFPE WES protocols.