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

Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, Lynch CF, Makhlouf H, McGregor P, McShane LM, Phillips Rohan J, Walsh WD, Williams PM, Gillanders EM, Mechanic LE, Schully SD - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0127353.g004: 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.

Mentions: For the remaining 53 unique specimens, average read depth was 112x, and was statistically significantly different among specimens by storage time (p<0.001; Table 4, Fig 4). Percent of target covered was also associated with storage time (p<0.001; Table 4). A ten-year difference in specimen age was associated with a 39x lower average read depth and 6% lower average percent target covered. Although, there is correlation of specimen age with read depth and target coverage, the results were still acceptable for data analysis. Of the 53 specimens, percent duplication increased from an average of 27% for specimens stored the shortest time to 40% for specimens stored the longest, indicating the uniquely mapped reads decreased from 73% to 60% over additional 10 years of sample storage time.


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

Carrick DM, Mehaffey MG, Sachs MC, Altekruse S, Camalier C, Chuaqui R, Cozen W, Das B, Hernandez BY, Lih CJ, Lynch CF, Makhlouf H, McGregor P, McShane LM, Phillips Rohan J, Walsh WD, Williams PM, Gillanders EM, Mechanic LE, Schully SD - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519244&req=5

pone.0127353.g004: 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.
Mentions: For the remaining 53 unique specimens, average read depth was 112x, and was statistically significantly different among specimens by storage time (p<0.001; Table 4, Fig 4). Percent of target covered was also associated with storage time (p<0.001; Table 4). A ten-year difference in specimen age was associated with a 39x lower average read depth and 6% lower average percent target covered. Although, there is correlation of specimen age with read depth and target coverage, the results were still acceptable for data analysis. Of the 53 specimens, percent duplication increased from an average of 27% for specimens stored the shortest time to 40% for specimens stored the longest, indicating the uniquely mapped reads decreased from 73% to 60% over additional 10 years of sample storage time.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Control and Population Sciences (DCCPS), National Cancer Institute, 9609 Medical Center Drive, Rockville, MD 20850, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus