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

Abstract

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.

Fig 1. Workflow for the FFPE tissues.

Three SEER RTRs selected cases that would likely meet the study criteria from their database. Cases were screened for histology/morphology, tumor nuclei content, and necrotic cell content to select cases meet the study criteria. The RTRs screened 30 or 31 cases; one RTR did not keep track of the number of cases they screened. Each RTR selected and sent 20 cases that met study criteria to the lab. One case was excluded from analyses due to incomplete information regarding storage time. 59 cases were considered for subsequent analyses. DNA was successfully extracted from 58 cases. WES was successful on 53 of the cases.

Fig 2. Association between specimen storage time and the Q129/41 ratio.

The solid line indicates the estimated linear relationship between age and Q129/41 ratio. The shaded area denotes pointwise 95% confidence intervals of the conditional mean. Cases successful through the entire WES workflow (DNA extraction through WES sequencing) are denoted as circles (N = 53); unsuccessful cases are denoted as X’s (N = 6).

Fig 3. Association between specimen storage time and final library size in base pairs.

Specimens that failed sequencing do not have library size values and are indicated by x. The solid line indicates the estimated linear relationship between storage time and library size (N = 59). The shaded area denotes pointwise 95% confidence intervals for the conditional means. Cases successful through the entire WES workflow (DNA extraction through WES sequencing) are denoted as circles (N = 53); unsuccessful cases are denoted as X’s (N = 6). Failed assays were not used to estimate the linear trend.

Fig 4. Association between specimen storage time and average read depth.

The solid line indicates the estimated linear relationship between age and average read depth. The shaded area denoted pointwise 95% confidence intervals of the conditional mean. Cases successful through the entire WES workflow (DNA extraction through WES sequencing) are denoted as circles (N = 53); unsuccessful cases are denoted as X’s (N = 6). Failed assays were not used to estimate the linear trend.