title: Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping.
abstract: There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000× can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels ≤12 bp long were detected by the variant caller software either exactly or as mismatched nucleotides within the indel region. The rapid turnaround time and low input DNA requirements make the multiplex PCR and semiconductor-based sequencing approach a viable option for mutation detection in a clinical laboratory.
authors.affiliation: Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Beadling
authors.forename: Tanaya L
authors.initials: TL
authors.lastname: Neff
authors.forename: Michael C
authors.initials: MC
authors.lastname: Heinrich
authors.forename: Katherine
authors.initials: K
authors.lastname: Rhodes
authors.forename: Michael
authors.initials: M
authors.lastname: Thornton
authors.forename: John
authors.initials: J
authors.lastname: Leamon
authors.forename: Mark
authors.initials: M
authors.lastname: Andersen
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Corless
mesh_terms: D015723:Gene Library; D014644:Genetic Variation; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D012372:ROC Curve; D012666:Semiconductors; D017422:Sequence Analysis, DNA