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Updated: Apr 4, 2026
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms
George J Burghel1, Carolyn D Hurst2, Christopher M Watson3
1Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds LS9 7TF, UK ; Leeds Institute of Cancer & Pathology, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK.
Abstract:
Detection of clinically actionable mutations in diagnostic tumour specimens aids in the selection of targeted therapeutics. With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis. As it is still not feasible for routine diagnostic laboratories to perform sequencing of the entire cancer genome, our approach was to undertake targeted mutation detection. To optimise our diagnostic workflow, we evaluated three target enrichment strategies using two next-generation sequencing (NGS) platforms (Illumina MiSeq and Ion PGM). The target enrichment strategies were Fluidigm Access Array custom amplicon panel including 13 genes (MiSeq sequencing), the Oxford Gene Technologies (OGT) SureSeq Solid Tumour hybridisation panel including 60 genes (MiSeq sequencing), and an Ion AmpliSeq Cancer Hotspot Panel including 50 genes (Ion PGM sequencing). DNA extracted from formalin-fixed paraffin-embedded (FFPE) blocks of eight previously characterised cancer cell lines was tested using the three panels. Matching genomic DNA from fresh cultures of these cell lines was also tested using the custom Fluidigm panel and the OGT SureSeq Solid Tumour panel. Each panel allowed mutation detection of core cancer genes including KRAS, BRAF, and EGFR. Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.
Insights
Targeted next-generation sequencing (NGS) panels accurately detect clinically actionable mutations in tumour DNA. This study optimized diagnostic workflows by evaluating three enrichment strategies for cancer gene panels, showing reliable variant detection from formalin-fixed paraffin-embedded (FFPE) samples.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Clinically actionable mutations in diagnostic tumor specimens guide targeted therapy selection.
- Tumor genetic diagnostics are shifting towards multigene analysis due to the increasing number of identified mutations.
- Whole-genome sequencing is not yet feasible for routine diagnostic laboratories, necessitating targeted mutation detection.
Purpose of the Study:
- To optimize diagnostic workflows for targeted mutation detection.
- To evaluate three distinct target enrichment strategies for next-generation sequencing (NGS) platforms.
- To assess the accuracy of variant detection using different gene panels and sequencing platforms, including from formalin-fixed paraffin-embedded (FFPE) DNA.
Main Methods:
- Three target enrichment strategies were evaluated: Fluidigm Access Array (13 genes, MiSeq), Oxford Gene Technologies (OGT) SureSeq Solid Tumour panel (60 genes, MiSeq), and Ion AmpliSeq Cancer Hotspot Panel (50 genes, Ion PGM).
- DNA from eight characterized cancer cell lines (formalin-fixed paraffin-embedded blocks and fresh cultures) was analyzed using the selected panels.
- Next-generation sequencing was performed on Illumina MiSeq and Ion PGM platforms.
Main Results:
- All evaluated panels successfully detected mutations in core cancer genes such as KRAS, BRAF, and EGFR.
- Accurate variant detection was achieved even when sequencing DNA extracted from formalin-fixed paraffin-embedded (FFPE) specimens.
- The study demonstrated the feasibility of using targeted gene panels for routine tumor genetic diagnostics.
Conclusions:
- Targeted mutation detection using NGS panels is a viable approach for clinical diagnostics.
- The evaluated enrichment strategies and NGS platforms provide accurate detection of actionable mutations from FFPE tumor samples.
- Optimized diagnostic workflows utilizing targeted gene panels can support the selection of targeted therapeutics in cancer care.
