Formalin fixation followed by paraffin embedding (FFPE) is the most widely used method of tissue preservation and often used in the clinical setting for cancer specimens. However, this process of fixation has negative consequences on the quality of extracted DNA, and inevitably adversely affects downstream analyses such as next generation sequencing (NGS). DNA extraction from FFPE samples requires paraffin disassociation, tissue rehydration and de-crosslinking. Low efficiency in any step will yield low quantities of nucleic acids with incomplete reversal of formaldehyde crosslinks, and mixtures of single and double stranded DNA. In addition, low quality nucleic acids will often result in poor sequencing quality due to inefficient library preparation, lower number of reads with high quality, and variable recovery of sequence reads with high GC-content.
Here we describe the use of Adaptive Focused Acoustics technology (Covaris Inc. 2013) to remove paraffin and break down tissue, and we compared this method to traditional xylene-based paraffin removal. We found that Covaris had superior performance overall: (1) DNA yields were 6 times higher, and less specimens failed extraction: this is critically important when dealing with small tumor biopsies. (2) DNA was more readily available for PCR amplification. (3) Mean coverage of DNA fragments with high GC content was higher and more reproducible. (4) Library complexity was higher. In addition, the Covaris protocol required less hands-on time in the lab, and eliminated the need for hazardous organic solvents for paraffin removal.
In conclusion, the Covaris FFPE DNA extraction method is very efficient, enhances DNA availability, improves reproducibility and provides higher DNA quality for NGS.
Citation Format: Ling Lin, Mikenah Vega, Robert T. Jones, Liuda Ziaugra, Deniz N. Dolcen, Ashwini Sunkavalli, Laura Schubert, Jack R. Lepine, Aaron R. Thorner, Matthew D. Ducar, William C. Hahn, Matthew L. Meyerson, Laura E. MacConaill, Paul Van Hummelen. Improved FFPE DNA extraction for next generation sequencing using adaptive focused acoustics technology. [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 4286. doi:10.1158/1538-7445.AM2014-4286
- ©2014 American Association for Cancer Research.