Use of FFPE-Derived DNA in Next Generation Sequencing: DNA extraction methods

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in NGS is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent ds DNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, whole exome (WES) and targeted sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES.