title: Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.
abstract: High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.
authors: Department of Ecology and Evolution, University of Lausanne, CH-1015 Lausanne, Switzerland. Alan.Brelsford@unil.ch
authors: Alan
authors: A
authors: Brelsford
authors: Hélène
authors: H
authors: Collin
authors: Nicolas
authors: N
authors: Perrin
authors: Luca
authors: L
authors: Fumagalli
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D000818:Animals; D003530:Cyprinidae / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015723:Gene Library; D012275:Oryza / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase