title: COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing.
abstract: BACKGROUND
  Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.
  
  
  METHODS
  We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.
  
  
  RESULTS
  In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.
  
  
  CONCLUSIONS
  As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: Coren A
authors.initials: CA
authors.lastname: Milbury
authors.forename: Mick
authors.initials: M
authors.lastname: Correll
authors.forename: John
authors.initials: J
authors.lastname: Quackenbush
authors.forename: Renee
authors.initials: R
authors.lastname: Rubio
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D001483:Base Sequence; D003110:Colonic Neoplasms / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D018773:Genes, erbB-1 / Q000235:genetics; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D004247:DNA; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins