LB11
Sensitive mutation detection in formalin fixed tumour samples by deep clonal sequencing
Neil Gibson, Hedley Carr, Jessica Whiteley, Gillian Ellison, John Morten
AstraZeneca, Cheshire, UK
Background
Due to their functional link to disease, somatic mutations are valuable markers of tumour development, disease progression and response to treatment. Mutation discovery in tumours by sequencing is therefore a high priority activity within cancer genetics.
Tumours contain both neoplastic and untransformed cells. Mutation discovery methods must be able to detect alleles arising in cancer genomes against a background of the normal allele. Capillary sequencing typically has a sensitivity in the range 20-50%. As many tumour samples contain <50% cancer cells this may result in non-detection of mutations.
The majority of tumour sample collections are formalin preserved, a process that damages DNA, therefore performing sensitive mutation discovery in heterogeneous, fixed tumours is a significant challenge.
The genetic profiling of these samples to discover biomarkers of disease progression and treatment selection will require new techniques with greater mutation detection sensitivity.
Polyclonal sequence data was generated from formalin fixed samples to determine whether clonal analysis would increase mutation detection sensitivity. In addition, we develop a new technique to perform DNA barcoding to enable efficient multiplex sequencing of PCR products from multiple DNA samples.
Method
Genomic DNA was amplified in duplicate using tagged primers and then re-amplified using barcoded primers unique to each sample. Products were combined to generate duplicate pools and sequenced using 454 sequencing.
Results
Sequencing Success
793/1152 (69%) of Sanger reads gave useable data.
1142/1152 (99%) of 454 targets gave useable data.
Mutation Detection Success
For 60.4% of targets it was possible to obtain >500 reads, equating to a
predicted sensitivity of ~5% clonal frequency (when observed in both pools).
Conclusion
Clonal sequencing of formalin fixed tumours enables mutation detection down to an estimated frequency of 5%. The DNA barcoding process is simple to apply and worked with Roche Diagnostics Amplicon software to unambiguously assign sequence data to the 24 individual samples.
