Robustness of Next Generation Sequencing on Older Formalin-Fixed Paraffin-Embedded Tissue.
Author(s): 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
Publication: PLoS One, 2015, Vol. 10, Page e0127353
PubMed ID: 26222067 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if the duration that formalin-fixed paraffin-embedded (FFPE) specimens are stored for impacts DNA yield, DNA quality, whole exome library construction, or the identification of mutations using whole exome sequencing.
Conclusion of Paper
Significant declines in DNA yield, DNA quality, average library fragment length, the percentage of targets with at least 20 x coverage, and average read depth were found with longer storage durations (3-32 years). The ratio of transition to transversion substitutions also increased with FFPE block storage. However, the success of whole exome sequencing was not affected. Importantly, 75% of FFPE specimens were found to have TP53 mutations, which the authors state is comparable to the 87% rate reported by the Cancer Genome Atlas (TCGA).
Studies
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Study Purpose
The purpose of this study was to determine if storage duration of FFPE specimens impacts DNA yield, DNA quality, whole exome library construction, or the identification of mutations using whole exome sequencing. A total of 59 ovarian serous adenocarcinoma specimens that had >50% of cells with nuclei consistent with malignancy and <50% necrotic cells were selected for analysis. The authors state that the storage conditions of the specimens were unknown and do not specify the timing of sectioning relative to DNA extraction. DNA and RNA were extracted from five 10 µm sections from each specimen using the Qiagen All Prep FFPET kit. DNA quality was measured using the real-time PCR amplification based Kapa Human Genomic DNA Quantification and QC Kit (KapaQC) which compares amplifiable copies of 41 bp, 129 bp and 305 bp fragments to determine the integrity of the DNA.
Summary of Findings:
Insufficient DNA was obtained from 1 specimen and of the 58 FFPE specimens analyzed, 15 (25.4%) had DNA of poor quality (Q129/41 < 0.1) and five (8.47%) were of very poor quality (Q129/41 <0.04). Average DNA yields were 3.8 µg, 4.6 µg and 1.7 µg for specimens stored 3-12, 13-22, and 23-32 years, respectively (p=0.003). Similarly, the Q129/49 ratio declined with FFPE block storage, decreasing from 0.22 and 0.24 in specimens stored for 3-12 and 13-22 years, respectively, to 0.13 in specimens stored 23-32 years (p<0.001). FFPE block storage duration did not affect the success of library construction, but the Wald test revealed that declines in average library fragment length (p=0.001), percentage of targets with 20 x coverage (p<0.001), and average read depth (p<0.001) were observed with longer storage durations. The ratio of transition to transversion substitutions (p<0.001) also increased with storage duration (3-32 years). Most of the changes were between specimens stored for 13-22 years and those stored for 23-32 years, rather than between specimens stored for 3-12 and 13-22 years. Importantly, 75% of the specimens were found to have TP53 mutations, which the authors state is comparable to the 87% rate reported by TCGA.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Next generation sequencing DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 3-12 years
13-22 years
23-32 years