Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping

Carol Beadling, Tanaya L. Neff, Michael C. Heinrich, Katherine Rhodes, Michael Thornton, John Leamon, Mark Andersen, Christopher L. Corless

Research output: Contribution to journalArticle

97 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)171-176
Number of pages6
JournalJournal of Molecular Diagnostics
Volume15
Issue number2
DOIs
StatePublished - Mar 1 2013

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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