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Sci Rep. 2015 Nov 13;5:16498. doi: 10.1038/srep16498.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

Author information

1
Faculty of Archaeology, Leiden University, Einsteinweg 2, 2333 CC, Leiden, the Netherlands.
2
Department of Anthropology, University of Oklahoma, Norman, OK, USA.
3
Center for Geogenetics, University of Copenhagen, Denmark.
4
Department of Preventive Dentistry, Academic Center for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, the Netherlands.
5
Department of Anthropology, University of Cape Town, South Africa.
6
Departament de Prehistòria i Arqueologia, Universitat de València, Spain.
7
Department of Human Evolution, Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
8
School of Social Sciences, Humanities, and Arts, University of California, Merced, USA.
9
Department of Archaeology, University of York, York, UK.
10
Department of Anthropology, University of British Columbia, Vancouver, Canada.
11
Department of Biology, University of York, York, UK.

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.

PMID:
26563586
PMCID:
PMC4643231
DOI:
10.1038/srep16498
[Indexed for MEDLINE]
Free PMC Article

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