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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 frequency detected using fresh genomic DNA. Comparison of mutant allele frequencies detected by the Fluidigm panel and the SureSeq panel.
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Related In: Results  -  Collection


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fig1: Mutant allele frequency detected using fresh genomic DNA. Comparison of mutant allele frequencies detected by the Fluidigm panel and the SureSeq panel.

Mentions: Using the SureSeq panel, all of the known mutations were detected and the two novel mutations identified by the Fluidigm custom panel were confirmed. The depth of coverage and mutant allele frequency of all of the variants are summarised in Supplementary Table 8. Figure 1 summarises the mutant allele frequency of the 24 mutations using the Fluidigm panel and the SureSeq panel. For 21 of 22 mutations detected successfully by both platforms, the mutant allele frequency was comparable. The TP53 mutation c.960G>C had a significantly different mutant allele frequency between the 2 platforms: 42.1% (SureSeq panel) and 99.7% (Fluidigm panel). Based on Sanger sequencing, the mutation appeared to be heterozygous (~50% mutant allele frequency). The depth of coverage and mutant allele frequency of the variants as detected by the Fluidigm and the SureSeq panels in fresh gDNA are summarised in Supplementary Table 8. Notably, three further variants (false positives) were identified in the SureSeq panel with <10% variant allele frequency (Table 2).


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 frequency detected using fresh genomic DNA. Comparison of mutant allele frequencies detected by the Fluidigm panel and the SureSeq panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Mutant allele frequency detected using fresh genomic DNA. Comparison of mutant allele frequencies detected by the Fluidigm panel and the SureSeq panel.
Mentions: Using the SureSeq panel, all of the known mutations were detected and the two novel mutations identified by the Fluidigm custom panel were confirmed. The depth of coverage and mutant allele frequency of all of the variants are summarised in Supplementary Table 8. Figure 1 summarises the mutant allele frequency of the 24 mutations using the Fluidigm panel and the SureSeq panel. For 21 of 22 mutations detected successfully by both platforms, the mutant allele frequency was comparable. The TP53 mutation c.960G>C had a significantly different mutant allele frequency between the 2 platforms: 42.1% (SureSeq panel) and 99.7% (Fluidigm panel). Based on Sanger sequencing, the mutation appeared to be heterozygous (~50% mutant allele frequency). The depth of coverage and mutant allele frequency of the variants as detected by the Fluidigm and the SureSeq panels in fresh gDNA are summarised in Supplementary Table 8. Notably, three further variants (false positives) were identified in the SureSeq panel with <10% variant allele frequency (Table 2).

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