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.
Highlights
To date, characterization of ancient oral and gut microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene
Following deep sequencing of 16S rRNA gene V3 amplicons on an Illumina MiSeq platform, we assigned sequences to Operational Taxonomic Units (OTUs) at 97% similarity using the closed-reference OTU protocol implemented in QIIME38,39, and the Greengenes 13.8 database[26] as a reference
We explored the impact that temperature history would have on the relative survival of the V3 regions of the taxa presented in Table 2 for a hypothetical oral microbiome sample averaged from all entries in the Human Oral Microbiome Database (HOMD)
Summary
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. 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 In practice this metataxonomic approach often produces highly skewed taxonomic frequency data. Recent studies of microbiome variation in humans[13,14,15,16,17] and non-human primates[9,18,19,20,21] have primarily characterized host-associated microorganisms using an amplicon metataxonomic approach[22], in which a targeted variable region of the 16S rRNA gene is amplified by polymerase chain reaction (PCR), deep sequenced using Generation Sequencing (NGS) technology, and compared to a reference database of 16S rRNA gene sequences. The relatively long length of the V4 region (approx. 292 bp, primer inclusive) makes it impractical for ancient DNA (aDNA) studies given that aDNA is known to be highly fragmented and rarely exceeds 200 bp in length[29]
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