title: Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR.
abstract: BACKGROUND
  Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-resequencing. Analysis of mutations below approximately 2%-5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids is problematic owing to increased noise from sequencing errors. Technologies that reduce noise via deep sequencing unavoidably reduce throughput and increase cost. Here we provide proof of principle that coamplification at lower denaturation temperature (COLD)-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput.
  
  
  METHODS
  We have developed a multiplex temperature-tolerant COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. After multiplex preamplification from genomic DNA, we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for melting temperature (Tm)-increasing mutations over all amplicons, in a single tube. Therefore, in separate reactions, both Tm-decreasing and Tm-increasing mutations are enriched.
  
  
  RESULTS
  Using custom-made and commercial gene panels containing 8, 50, 190, or 16 000 amplicons, we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of deoxyinosine triphosphate (dITP)/2,6-diaminopurine triphosphate (dDTP) in place of deoxyguanosine triphosphate (dGTP)/deoxyadenosine triphosphate (dATP) enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit of detection of approximately 0.01%-0.1% mutation abundance by use of Ion-Torrent and 0.1%-0.3% by use of Sanger sequencing.
  
  
  CONCLUSIONS
  Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single-tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.
authors.affiliation: Division of DNA Repair and Genome Stability and.
authors.forename: Elena
authors.initials: E
authors.lastname: Castellanos-Rizaldos
authors.affiliation: Transgenomic Inc., Omaha, NE.
authors.forename: Katherine
authors.initials: K
authors.lastname: Richardson
authors.affiliation: Transgenomic Inc., Omaha, NE.
authors.forename: Rui
authors.initials: R
authors.lastname: Lin
authors.affiliation: Transgenomic Inc., Omaha, NE.
authors.forename: Grant
authors.initials: G
authors.lastname: Wu
authors.affiliation: Division of DNA Repair and Genome Stability and Division of Medical Physics and Biophysics, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; mmakrigiorgos@lroc.harvard.edu.
authors.forename: Mike G
authors.initials: MG
authors.lastname: Makrigiorgos
mesh_terms: D002460:Cell Line; D004247:DNA / Q000097:blood / Q000235:genetics*; D052138:Genes, Neoplasm; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009154:Mutation; D009369:Neoplasms / Q000097:blood / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D004247:DNA