Limits...
Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms.

Burghel GJ, Hurst CD, Watson CM, Chambers PA, Dickinson H, Roberts P, Knowles MA - Biomed Res Int (2015)

Bottom Line: With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis.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.Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire 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.

No MeSH data available.


Related in: MedlinePlus

Mutant allele frequencies detected in genomic DNA extracted from FFPE samples. Comparison of mutant allele frequencies detected by the Fluidigm panel, the SureSeq panel, and the Ion AmpliSeq panel.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4553307&req=5

fig2: Mutant allele frequencies detected in genomic DNA extracted from FFPE samples. Comparison of mutant allele frequencies detected by the Fluidigm panel, the SureSeq panel, and the Ion AmpliSeq panel.

Mentions: Using the Ion AmpliSeq panel, 18 of the 24 mutations were detected. The 6 missed mutations were in TP53 (c.120G>A, c.221_236del16, c.338T>G, c.670G>A, c.783-2A>G, and c.960G>C), which are not among the hotspot mutations detected by this panel. The depth of coverage and mutant allele frequency of all of the variants are summarised in Supplementary Table 9. Figure 2 summarises the mutant allele frequency of the 24 mutations using the 3 panels.


Towards a Next-Generation Sequencing Diagnostic Service for Tumour Genotyping: A Comparison of Panels and Platforms.

Burghel GJ, Hurst CD, Watson CM, Chambers PA, Dickinson H, Roberts P, Knowles MA - Biomed Res Int (2015)

Mutant allele frequencies detected in genomic DNA extracted from FFPE samples. Comparison of mutant allele frequencies detected by the Fluidigm panel, the SureSeq panel, and the Ion AmpliSeq panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4553307&req=5

fig2: Mutant allele frequencies detected in genomic DNA extracted from FFPE samples. Comparison of mutant allele frequencies detected by the Fluidigm panel, the SureSeq panel, and the Ion AmpliSeq panel.
Mentions: Using the Ion AmpliSeq panel, 18 of the 24 mutations were detected. The 6 missed mutations were in TP53 (c.120G>A, c.221_236del16, c.338T>G, c.670G>A, c.783-2A>G, and c.960G>C), which are not among the hotspot mutations detected by this panel. The depth of coverage and mutant allele frequency of all of the variants are summarised in Supplementary Table 9. Figure 2 summarises the mutant allele frequency of the 24 mutations using the 3 panels.

Bottom Line: With an ever increasing number of clinically significant mutations identified, tumour genetic diagnostics is moving from single to multigene analysis.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.Our results indicate that the panels enable accurate variant detection despite sequencing from FFPE DNA.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire 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.

No MeSH data available.


Related in: MedlinePlus