title: AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities.
abstract: High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches ("AmpliconDuo filter"). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.
authors.affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Lange
authors.affiliation: Department of Biodiversity, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Jost
authors.affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Heider
authors.affiliation: Department of Biodiversity, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Bock
authors.affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Budeus
authors.affiliation: Eurofins Genomics, Ebersberg, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Schilling
authors.affiliation: Eurofins Genomics, Ebersberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Strittmatter
authors.affiliation: Department of Biodiversity, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Boenigk
authors.affiliation: Research Group Bioinformatics, Faculty of Biology, University of Duisburg-Essen, Essen, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoffmann
mesh_terms: D000465:Algorithms; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D064307:Microbiota / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*