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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 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).
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pone.0127353.g002: 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).

Mentions: All tissue curls were received in good condition at the MoCha lab. The NCI-conducted pathology review verified the majority of tissues met desired selection criteria (Table 2). Of 59 unique specimens, one sample yielded very minimal DNA, and was considered to have failed DNA extraction; however, the Q129/41 ratio was used for age group correlation. An average of 3.7μg DNA was observed in the 58 samples that yielded DNA. Fifteen of 59 tissues (25.4%) had poor DNA quality as determined by the KapaQC result (Q129/41<0.1), while 8.47% had very poor DNA quality (Q129/41<0.04) (Table 3, Fig 2).


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 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0127353.g002: 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).
Mentions: All tissue curls were received in good condition at the MoCha lab. The NCI-conducted pathology review verified the majority of tissues met desired selection criteria (Table 2). Of 59 unique specimens, one sample yielded very minimal DNA, and was considered to have failed DNA extraction; however, the Q129/41 ratio was used for age group correlation. An average of 3.7μg DNA was observed in the 58 samples that yielded DNA. Fifteen of 59 tissues (25.4%) had poor DNA quality as determined by the KapaQC result (Q129/41<0.1), while 8.47% had very poor DNA quality (Q129/41<0.04) (Table 3, Fig 2).

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