title: Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies.
abstract: Amplicon-based marker gene surveys form the basis of most microbiome and other microbial community studies. Such PCR-based methods have multiple steps, each of which is susceptible to error and bias. Variance in results has also arisen through the use of multiple methods of next-generation sequencing (NGS) amplicon library preparation. Here we formally characterized errors and biases by comparing different methods of amplicon-based NGS library preparation. Using mock community standards, we analyzed the amplification process to reveal insights into sources of experimental error and bias in amplicon-based microbial community and microbiome experiments. We present a method that improves on the current best practices and enables the detection of taxonomic groups that often go undetected with existing methods.
authors.affiliation: University of Minnesota Genomics Center, Minneapolis, Minnesota, USA.
authors.forename: Daryl M
authors.identifier: 0000-0002-4434-2788
authors.initials: DM
authors.lastname: Gohl
authors.affiliation: Biomedical Informatics and Computational Biology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Pajau
authors.initials: P
authors.lastname: Vangay
authors.affiliation: Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: John
authors.initials: J
authors.lastname: Garbe
authors.affiliation: University of Minnesota Genomics Center, Minneapolis, Minnesota, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: MacLean
authors.affiliation: University of Minnesota Genomics Center, Minneapolis, Minnesota, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Hauge
authors.affiliation: University of Minnesota Genomics Center, Minneapolis, Minnesota, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Becker
authors.affiliation: University of Minnesota Informatics Institute, Minneapolis, Minnesota, USA.
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Gould
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Clayton
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Johnson
authors.affiliation: Department of Microbiology and Immunology, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Hunter
authors.affiliation: Biotechnology Institute, University of Minnesota, St. Paul, Minnesota, USA.|Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Knights
authors.affiliation: University of Minnesota Genomics Center, Minneapolis, Minnesota, USA.
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Beckman
mesh_terms: D004261:DNA Replication / Q000235:genetics*; D015723:Gene Library; D005819:Genetic Markers / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D064307:Microbiota / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D017410:Practice Guidelines as Topic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005819:Genetic Markers