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PLoS One. 2015 Jul 29;10(7):e0127353. doi: 10.1371/journal.pone.0127353. eCollection 2015.

Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.

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Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.
Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc. and Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States of America.
Division of Cancer Treatment and Diagnosis (DCTD), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.
USC Keck School of Medicine, University of Southern California, 1441 Eastlake Ave. NOR 4451A, 9175 Los Angeles, CA 90089-9175, United States of America.
University of Hawaii Cancer Center, University of Hawaii, 701 Ilalo Street Honolulu, HI 96813, United States of America.
Department of Epidemiology, College of Public Health, 145 North Riverside Dr., The University of Iowa, Iowa City, IA 52242, United States of America.


Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

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