title: Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results.
abstract: Library preparation protocols for high-throughput DNA sequencing (HTS) include amplification steps in which errors can build up. In order to have confidence in the sequencing data, it is important to understand the effects of different Taq polymerases and PCR amplification protocols on the DNA molecules sequenced. We compared thirteen enzymes in three different marker systems: simple, single copy nuclear gene and complex multi-gene family. We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps. We find that enzyme choice has a large impact on the proportion of correct sequences recovered. The most complex marker systems yielded fewer correct reads, and the proportion of correct reads was greatly affected by the enzyme used. Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads. Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles. Consequently, enzyme selection for downstream HTS has important consequences, especially in complex genetic systems.
authors.affiliation: 1] Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain [2] Facultad de Medicina Veterinaria, Universidad de Panamá, Panama.
authors.forename: Claudia
authors.initials: C
authors.lastname: Brandariz-Fontes
authors.affiliation: Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Camacho-Sanchez
authors.affiliation: Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain.
authors.forename: Carles
authors.initials: C
authors.lastname: Vilà
authors.affiliation: Laboratorio de Investigación Aplicada, Cría Caballar de las Fuerzas Armadas, Córdoba, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Vega-Pla
authors.affiliation: University of the South Pacific, School of Marine Studies, Faculty of Science, Technology &Environment, Laucala Campus, Suva, Fiji.
authors.forename: Ciro
authors.initials: C
authors.lastname: Rico
authors.affiliation: Conservation and Evolutionary Genetics Group, Estación Biológica de Doñana (EBD-CSIC), Sevilla, Spain.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Leonard
mesh_terms: D000483:Alleles; D000818:Animals; D004247:DNA / Q000032:analysis*; D004272:DNA, Mitochondrial / Q000032:analysis; D059014:High-Throughput Nucleotide Sequencing; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism*; D019596:Wolves
chemical_list: D004272:DNA, Mitochondrial; D004247:DNA; D019914:Taq Polymerase