Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies.
Author(s): Sah S, Chen L, Houghton J, Kemppainen J, Marko AC, Zeigler R, Latham GJ
Publication: Genome Med, 2013, Vol. 5, Page 77
PubMed ID: 24001039 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare different methods of DNA quantification and optimize the real-time PCR based quantitative functional index (QFI) to allow for reliable identification of mutations in FFPE tissues using targeted next-generation sequencing (NGS).
Conclusion of Paper
The quantified yield of DNA was 17-fold higher by NanoDrop than by Qubit. The authors report that using QFI score to adjust the nanogram amount of DNA for NGS analysis can ensure sufficient amplifiable template, and screening specimens using a QFI threshold of >6% can reduced the incidence of false negatives and false positives.
Studies
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Study Purpose
The purpose of this study was to compare quantification methods for DNA extracted from FFPE specimens. A total of 165 FFPE specimens that were commercially obtained and stored for 1-18 years prior to analysis were used. FFPE specimens included 10 biopsy specimens each from ovary, colon, skin, breast and lung purchased from the Asuragen FFPE database, 76 FFPE thyroid biopsies purchased from Asterand, and 39 colorectal tumor resections purchased from Folio. DNA was extracted from all specimens using the RecoverAll FFPE kit. Each DNA sample was quantified using a spectrophotometer (Nanodrop), a DNA-binding fluorescent dye (Qubit), and a qPCR-based assay (QFI) that corresponds to the percentage of functional template for a given target.
Summary of Findings:
DNA amounts varied greatly among quantification methods, as the same specimen set resulted in a mean DNA yield of 5 ng with Nanodrop and 0.30 ng with Qubit, a 17-fold difference. Differences in the percentage of samples below the threshold of detection also differed between Qubit and QFI, as 19.4 and 6.1% of specimens failed to generate data, respectively. For specimens with a QFI score >3% there was a modest linear correlation between relative DNA concentration determined by Qubit and QFI score (R=0.66), while no correlation was observed for specimens with lower QFI scores.
Biospecimens
- Tissue - Breast
- Tissue - Ovary
- Tissue - Thyroid Gland
- Tissue - Skin
- Tissue - Lung
- Tissue - Colorectal
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Spectrophotometry DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Spectrophotometry Specific Technology platform Real-time PCR
Qubit
NanoDrop
Spectrophotometry Specific Quality metrics Determined by Qubit
Determined by QFI
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Study Purpose
The purpose of this study was to investigate the effects of re extraction, block storage, and amplicon choice on the QFI score calculated using real-time PCR data. The authors also explored whether the accuracy of NGS data generated with DNA from FFPE specimens could be improved if QFI was used to calculate the number of amplifiable templates. A total of 165 FFPE specimens that were commercially obtained and stored for 1-18 years prior to analysis were used. FFPE specimens included 10 biopsy specimens each from ovary, colon, skin, breast and lung purchased from the Asuragen FFPE database, 76 FFPE thyroid biopsies purchased from Asterand, and 39 colorectal tumor resections purchased from Folio. DNA was extracted from all specimens using the RecoverAll FFPE kit.
Summary of Findings:
In a subset of 32 specimens with a wide range of QFI scores (0.03-24.5%), no evidence of PCR inhibition was observed for any specimen. The authors report QFI was not affected by FFPE block storage or reextraction of specimens from the same block, nor was QFI dependent on the gene used for calculation as the QFI were comparable (average fold change =1.01) when calculated based on either tata-binding protein (TBP) or ferritin heavy polypeptide 1 (FTH1). However, the average QFI did differ among specimens from the Asuragen, Asterand and Folio Biosciences cohorts (average QFIs of 8.0, 6.0 and 3.5, respectively). The authors report that QFI scores can be successfully used to calculate the minimal number of amplifiable templates necessary for NGS-based mutation detection, and conclude that the incidence of false-negatives can be reduced when the nanogram amount of DNA used is adjusted accordingly. When DNA input amounts were adjusted based on QFI, an additional 2 mutations were detected than when QFI was not considered. The incidence of false positives was also influenced by a specimen's QFI score. Targeted NGS of 44 specimens resulted in more mutations in specimens with a QFI <3% than in specimens with a QFI >6% (166 versus 14.7, p=0.006). While 90% of variants identified in specimens with a QFI >6% were confirmed by a second targeted NGS method, only 44% of the variants identified with a QFI of <3% were confirmed using a second targeted NGS method.
Biospecimens
- Tissue - Ovary
- Tissue - Colorectal
- Tissue - Thyroid Gland
- Tissue - Breast
- Tissue - Skin
- Tissue - Lung
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Not specified
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Targeted nucleic acid TBP
FTH-1
Next generation sequencing Specific Template/input amount Unadjusted
Adjusted for QFI
Storage Storage duration 1-18 years