title: Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.
abstract: To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.
authors.forename: Kirsten A
authors.initials: KA
authors.lastname: Ziesemer
authors.affiliation: Department of Anthropology, University of Oklahoma, Norman, OK, USA.
authors.forename: Allison E
authors.initials: AE
authors.lastname: Mann
authors.affiliation: Department of Anthropology, University of Oklahoma, Norman, OK, USA.
authors.forename: Krithivasan
authors.initials: K
authors.lastname: Sankaranarayanan
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.|Center for Geogenetics, University of Copenhagen, Denmark.
authors.forename: Hannes
authors.initials: H
authors.lastname: Schroeder
authors.affiliation: Department of Anthropology, University of Oklahoma, Norman, OK, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Ozga
authors.affiliation: Department of Preventive Dentistry, Academic Center for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, the Netherlands.
authors.forename: Bernd W
authors.initials: BW
authors.lastname: Brandt
authors.affiliation: Department of Preventive Dentistry, Academic Center for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, the Netherlands.
authors.forename: Egija
authors.initials: E
authors.lastname: Zaura
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.
authors.forename: Andrea
authors.initials: A
authors.lastname: Waters-Rist
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.
authors.forename: Menno
authors.initials: M
authors.lastname: Hoogland
authors.affiliation: Department of Anthropology, University of Cape Town, South Africa.|Departament de Prehistòria i Arqueologia, Universitat de València, Spain.|Department of Human Evolution, Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
authors.forename: Domingo C
authors.initials: DC
authors.lastname: Salazar-García
authors.affiliation: School of Social Sciences, Humanities, and Arts, University of California, Merced, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Aldenderfer
authors.affiliation: Department of Archaeology, University of York, York, UK.
authors.forename: Camilla
authors.initials: C
authors.lastname: Speller
authors.affiliation: Department of Archaeology, University of York, York, UK.
authors.forename: Jessica
authors.initials: J
authors.lastname: Hendy
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.|Department of Anthropology, University of British Columbia, Vancouver, Canada.
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Weston
authors.affiliation: Department of Biology, University of York, York, UK.
authors.forename: Sandy J
authors.initials: SJ
authors.lastname: MacDonald
authors.affiliation: Department of Biology, University of York, York, UK.
authors.forename: Gavin H
authors.initials: GH
authors.lastname: Thomas
authors.affiliation: Department of Archaeology, University of York, York, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Collins
authors.affiliation: Department of Anthropology, University of Oklahoma, Norman, OK, USA.
authors.forename: Cecil M
authors.initials: CM
authors.lastname: Lewis
authors.affiliation: Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.
authors.forename: Corinne
authors.initials: C
authors.lastname: Hofman
authors.affiliation: Department of Anthropology, University of Oklahoma, Norman, OK, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Warinner
mesh_terms: D001106:Archaeology; D001419:Bacteria / Q000145:classification / Q000235:genetics; D003728:Dental Calculus / Q000382:microbiology; D005260:Female; D000069196:Gastrointestinal Microbiome / Q000235:genetics; D005784:Gene Amplification; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D054892:Metagenome / Q000235:genetics*; D056186:Metagenomics / Q000379:methods*; D041581:Methanobrevibacter / Q000145:classification / Q000235:genetics; D064307:Microbiota / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*
chemical_list: D012336:RNA, Ribosomal, 16S