A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology.
Author(s): Bettoni F, Koyama FC, de Avelar Carpinetti P, Galante PAF, Camargo AA, Asprino PF
Publication: Exp Mol Pathol, 2017, Vol. 103, Page 294-299
PubMed ID: 29175301 PubMed Review Paper? No
Purpose of Paper
This paper investigated if the percentage of DNA allowing for amplification of a 254 bp product was an indicator of NGS library yield and number of artifactual mutations detected.
Conclusion of Paper
The percentage of amplifiable DNA allowing for amplification of a 254 bp product was a good indicator of the library yield and number of false positive mutations. Using a cut-off of 5%, all specimens yielded libraries of the desired quantity for hybridization (>750 ng) and no false positive mutations, but when a cut-off of 0.25% was used library yields from some specimens were only acceptable (>350 ng) and there were some false positive mutations and below 0.25% the library yields were lower and the number of false positive mutations increased. Amplification of a 78 bp fragment misclassified some specimens in the acceptable and poor categories.
Studies
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Study Purpose
This study investigated if the percentage of DNA allowing for amplification of a 254 bp product was an indicator of NGS library yield and number of artifactual mutations detected. Tumor regions were microdissected from four to ten unstained 5 µm sections of 12 FFPE tumors (2 breast, 4 colon, 3 lung, 1 pancreas, 1 uterus, and 1 thyroid). No details of specimen processing or storage were provided. DNA was extracted from specimens of at least 25 mm3 using the GeneRead DNA FFPE kit. DNA concentrations were determined by fluorometry and DNA quality by real-time PCR amplification of intronic regions of MLH1. NGS libraries were constructed from 200 ng DNA using the Agilent Custom SureSelect capture kit and were assessed by fluorometry and bioanalyzer and sequenced on a MiSeq. Some specimens were also sequenced by Sanger sequencing.
Summary of Findings:
Library yield varied greatly among specimens but did not seem to reflect the tissue type or DNA extraction yield. Amplification efficiency of the 254 bp fragment was strongly correlated with library yield (ρ=0.86, P=0.0007). The recommended library yield of >750 ng was obtained only from the four specimens in which more than 5% of the 254 bp fragments were amplifiable and the acceptable library yield of 350 ng was obtained only from the six specimens from which 0.25% to 5% of 254 bp fragments were amplifiable. The number of false positive mutation calls increased with decreasing amplification efficiency of the 254 bp fragment. When the percentage of amplifiable 254 bp products dropped from 0.254% to 0.0001%, the number of mutations identified increased from nine to 254. The majority of the mutations discovered in the low quality specimens were C:G > T:A or C:G > A:T indicating deamination of cysteine or shearing in the presence of contaminants, respectively. Of the 296 mutations found when 0.001% of the 254 bp product was amplifiable, six were resequenced and only one was validated. In contrast, all 16 tested mutations identified in specimens in which greater than 5% of the 254 bp product was amplifiable were confirmed by Sanger sequencing. The percentage of amplifiable DNA of 78 bp did not work as well and misclassified some specimens in the acceptable and poor categories.
Biospecimens
- Tissue - Breast
- Tissue - Colorectal
- Tissue - Thyroid Gland
- Tissue - Pancreas
- Tissue - Uterus
- Tissue - Lung
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Next generation sequencing DNA DNA sequencing DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Next generation sequencing Specific Technology platform NGS
Sanger sequencing
Real-time qPCR Specific Length of gene fragment 78 bp
254 bp