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

<p>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).</p

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

<div><p>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.</p></div

Success of whole exome assays by SEER Registry sites.

<p>* Sequencing assay success defined as having a non-failed final library size and percent target coverage of 50% with a minimum of 20x coverage.</p><p>The p-value for a linear test for trend of success probability by site equals 0.693.</p><p>Success of whole exome assays by SEER Registry sites.</p

Whole Exome Sequencing QC metrics by duration of specimen storage.

<p><b>Final Library Size (bp):</b> This QC metric represents the peak size from BioAnalyzer electropherogram traces of the final exome library from each sample using the Agilent BioAnalyzer High Sensitivity DNA kit.</p><p><b>% Target Covered at 20x:</b> The percentage of all target bases achieving 20X or greater read depth. This metric measures the efficiency of the exome capture. DNA samples with poor quality samples tend to have lower % target covered.</p><p><b>Average Read Depth:</b> Average read depth in the target region.</p><p><b>Percent Duplication:</b> Percent of reads originating from same fragment of the library. These duplicated reads may indicate bias originating from sample quality, library amplification etc. DNA samples with poor quality samples tend to have higher percent duplication.</p><p><b>Ti/Tv Ratio:</b> This is ratio of transitions (single nucleotide substitutions with the same type of nucleotide, e.g., pyrimidine to pyrimidine (C<>T) or purine to purine (A<>G)) to transversions (single nucleotide substitutions with the different type of nucleotide, i.e., pyrimidine to purine or vice versa (A<>T etc.). FFPE DNA samples tend to have higher Ti/Tv ratio due to chemical crosslink and modification.</p><p>CI = confidence Interval. Overall means and standard deviations for quality measures and by storage time, differences per 10 years of storage and 95% confidence intervals estimated by linear regression on continuous time. P-values are for Wald tests of time coefficients in regression models. A slope of zero indicates no association with specimen storage time. P-values are not adjusted for multiple comparisons.</p><p>Whole Exome Sequencing QC metrics by duration of specimen storage.</p

Whole Exome Sequencing QC metrics by duration of specimen storage.

<p><b>Final Library Size (bp):</b> This QC metric represents the peak size from BioAnalyzer electropherogram traces of the final exome library from each sample using the Agilent BioAnalyzer High Sensitivity DNA kit.</p><p><b>% Target Covered at 20x:</b> The percentage of all target bases achieving 20X or greater read depth. This metric measures the efficiency of the exome capture. DNA samples with poor quality samples tend to have lower % target covered.</p><p><b>Average Read Depth:</b> Average read depth in the target region.</p><p><b>Percent Duplication:</b> Percent of reads originating from same fragment of the library. These duplicated reads may indicate bias originating from sample quality, library amplification etc. DNA samples with poor quality samples tend to have higher percent duplication.</p><p><b>Ti/Tv Ratio:</b> This is ratio of transitions (single nucleotide substitutions with the same type of nucleotide, e.g., pyrimidine to pyrimidine (C<>T) or purine to purine (A<>G)) to transversions (single nucleotide substitutions with the different type of nucleotide, i.e., pyrimidine to purine or vice versa (A<>T etc.). FFPE DNA samples tend to have higher Ti/Tv ratio due to chemical crosslink and modification.</p><p>CI = confidence Interval. Overall means and standard deviations for quality measures and by storage time, differences per 10 years of storage and 95% confidence intervals estimated by linear regression on continuous time. P-values are for Wald tests of time coefficients in regression models. A slope of zero indicates no association with specimen storage time. P-values are not adjusted for multiple comparisons.</p><p>Whole Exome Sequencing QC metrics by duration of specimen storage.</p

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

<p>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).</p

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

<p>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.</p

DNA quantity and quality metrics by storage time of specimen.

<p><b>DNA yield (μg) (measured by Qubit):</b> Extracted DNA concentration was measured using Qubit fluorometer using dsDNA BR Assay Kits from Life Technologies Inc. and total DNA yield for the sample were calculated.</p><p><b>A260/280:</b> Absorbance at 260 nm and 280 nm were measured using Nanodrop for extracted DNA from each sample and expressed as a ratio.</p><p><b>KapaQC Q129/41 ratio:</b> Kapa Human Genomic DNA Quantification and QC Kit [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127353#pone.0127353.ref023" target="_blank">23</a>] was used to measure the quality of the genomic DNA isolated from these FFPE samples. This ratio is a measure of fragmentation of the genomic DNA extracted from the sample.</p><p>*CI = confidence Interval. Overall means and standard deviations for quality measures and by storage time, differences per 10 years of storage and 95% confidence intervals estimated by linear regression on continuous time. P-values are for Wald tests of time coefficients in regression models. A slope of zero indicates no association with specimen storage time. P-values are not adjusted for multiple comparisons.</p><p>^$ 1 sample, age group 13–22 years, is missing DNA yield because no DNA remained after the QC step.</p><p>DNA quantity and quality metrics by storage time of specimen.</p

Association between specimen storage time and average read depth.

<p>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.</p

FFPE specimens selected for analysis.

<p>*For seven cases two separate specimens were prepared and sent to the laboratory.</p><p>FFPE specimens selected for analysis.</p