title: Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples.
abstract: Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.
authors.affiliation: Genetics, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena, 30202, Cartagena, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Pawluczyk
authors.forename: Julia
authors.initials: J
authors.lastname: Weiss
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Links
authors.forename: Mikel
authors.initials: M
authors.lastname: Egaña Aranguren
authors.forename: Mark D
authors.initials: MD
authors.lastname: Wilkinson
authors.forename: Marcos
authors.initials: M
authors.lastname: Egea-Cortines
mesh_terms: D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*