title: False positives in multiplex PCR-based next-generation sequencing have unique signatures.
abstract: Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Chad M
authors.initials: CM
authors.lastname: McCall
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Stacy
authors.initials: S
authors.lastname: Mosier
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Michele
authors.initials: M
authors.lastname: Thiess
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Marija
authors.initials: M
authors.lastname: Debeljak
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Aparna
authors.initials: A
authors.lastname: Pallavajjala
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Katie
authors.initials: K
authors.lastname: Beierl
authors.affiliation: Department of Pathology, Duke University School of Medicine, Durham, North Carolina.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Deak
authors.affiliation: Department of Pathology, Duke University School of Medicine, Durham, North Carolina.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Datto
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland. Electronic address: jeshlem@jhmi.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D000465:Algorithms; D019295:Computational Biology / Q000379:methods; D066246:ErbB Receptors / Q000235:genetics; D005091:Exons; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D066246:ErbB Receptors