Back to Search
Start Over
Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material
- Source :
- PLoS One (print), 11(2):e0149405. Public Library of Science, PLoS ONE [E], 11(2). Public Library of Science, PLoS ONE, Vol 11, Iss 2, p e0149405 (2016), PLoS ONE
- Publication Year :
- 2016
-
Abstract
- textabstractBackground Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5%concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. Conclusion Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.
- Subjects :
- 0301 basic medicine
Tissue Fixation
Molecular biology
Somatic cell
Mutagenesis and Gene Deletion Techniques
DNA Mutational Analysis
Gene Identification and Analysis
Gene Sequencing
lcsh:Medicine
Biochemistry
chemistry.chemical_compound
Sequencing techniques
0302 clinical medicine
Neoplasms
Medicine and Health Sciences
DNA sequencing
lcsh:Science
Non-U.S. Gov't
Genetics
Medicine(all)
Paraffin Embedding
Multidisciplinary
Agricultural and Biological Sciences(all)
Research Support, Non-U.S. Gov't
High-Throughput Nucleotide Sequencing
Genomics
Cancer treatment
Oncology
030220 oncology & carcinogenesis
Transcriptome Analysis
Research Article
Next-Generation Sequencing
Formalin fixed paraffin embedded
Biology
Biomolecular isolation
Research Support
03 medical and health sciences
Diagnostic Medicine
Formaldehyde
Cancer Detection and Diagnosis
Journal Article
Humans
Mutation Detection
Biology and life sciences
Biochemistry, Genetics and Molecular Biology(all)
lcsh:R
Computational Biology
Reproducibility of Results
Ion semiconductor sequencing
Genome Analysis
DNA isolation
DNA extraction
Research and analysis methods
Molecular biology techniques
Mutational Analysis
030104 developmental biology
chemistry
Mutation
lcsh:Q
DNA
Genetics and Molecular Biology(all)
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 11
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- PLoS ONE [E]
- Accession number :
- edsair.doi.dedup.....c26df4c8ff3a2917dfacabe1e2a66c71