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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 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.
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pone.0127353.g003: 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.

Mentions: After completion of the library preparation protocol, two of the 58 specimens (3.4%) did not have detectable DNA at the final quantification step; one was in the 3 to 12 and one in the 23 to 32 storage-year categories. Three specimens had very low final library concentrations (<1nM); one was in the 13 to 22 and two in the 23 to 32 storage-year categories. However, those five specimens were sequenced to determine if there was any information to be collected to set the thresholds for specimens with the lowest acceptable quality. The average fragment size in final libraries was associated with specimen storage time (ten-year difference in specimen age was associated with an approximately 9bp lower library; Fig 3 and Table 4).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127353.g003: 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.
Mentions: After completion of the library preparation protocol, two of the 58 specimens (3.4%) did not have detectable DNA at the final quantification step; one was in the 3 to 12 and one in the 23 to 32 storage-year categories. Three specimens had very low final library concentrations (<1nM); one was in the 13 to 22 and two in the 23 to 32 storage-year categories. However, those five specimens were sequenced to determine if there was any information to be collected to set the thresholds for specimens with the lowest acceptable quality. The average fragment size in final libraries was associated with specimen storage time (ten-year difference in specimen age was associated with an approximately 9bp lower library; Fig 3 and Table 4).

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