Abstract 3497: A novel method of active paraffin removal and efficient extraction of NGS-quality DNA from FFPE tissues

Abstract

Abstract Formalin Fixation and Paraffin Embedding (FFPE) of tissues remains a routine histology practice by pathologists due to the quality and stability of structural information preserved. The method was conceived primarily to facilitate histological analysis rather than molecular approaches increasingly common in clinical oncology settings such as targeted sequencing and whole genome sequencing. Chemical crosslinks and the paraffin matrix of FFPE-preserved samples present significant challenges for the robust extraction of nucleic acids. To address this and provide reliable access to clinical samples, we present a novel method for consistent and reproducible extraction and purification of DNA from FFPE samples using Covaris Adaptive Focused Acoustics (AFA). Use of focused ultrasonic energy to emulsify embedding paraffin. The method is predicated on the use of highly controlled acoustic energy to efficiently remove paraffin from FFPE cores, sections, and slides enabling efficient tissue rehydration, tissue digestion, crosslink reversal, and nucleic acid release. In addition, this critical step is carried out without the use of organic solvents or mineral oils. We present quantitative fluorescent microscopy illustrating the efficiency of paraffin removal from tissues utilizing this novel approach. Better data quality as a consequence of improved DNA yield and quality. Having established reproducible sample processing we sought to explore benefits in downstream applications and analyses. Results, utilizing replicates of several different tissue types, indicate high dsDNA yields, controllable fragment sizes and excellent DNA quality as indicated by commercial qPCR based kits designed to assess DNA quality. Improved sequencing coverage as a consequence of improved DNA yield and quality. The quality of the extracted DNA, as a function of consistent sequence coverage across the entire genome, was then confirmed using whole genome sequencing of the FFPE samples as compared to sequence data obtained from matched fresh frozen tissue. Our data also indicate that the high quality controllable DNA extraction allows for preparation of large insert (1-6kb) libraries previously considered very difficult from FFPE stabilized tissues. Method suitable for automation. The simple workflow allows for the parallel processing of up to 8 FFPE samples in a 96 well plate format. We present data showing highly reproducible results while processing a large number of FFPE samples. Citation Format: Hamid Khoja, Edwin Rudd, Austin Purdy, James Han, Srikanth Kakumanu, Adrian Palmer, Guillaume Durin, Jim Laugharn. A novel method of active paraffin removal and efficient extraction of NGS-quality DNA from FFPE tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2014-3497