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

Ziesemer, Kirsten A., Mann, Allison E., Sankaranarayanan, Krithivasan, Schroeder, Hannes, Ozga, Andrew T., Brandt, Bernd W., Zaura, Egija, Waters-Rist, Andrea, Hoogland, Menno, Salazar-García, Domingo C., Aldenderfer, Mark, Speller, Camilla, Hendy, Jessica, Weston, Darlene A., MacDonald, Sandy J., Thomas, Gavin H., Collins, Matthew J., Lewis, Cecil M., Hofman, Corinne, Warinner, Christina·2015·Scientific Reports
DOI 10.1038/srep16498PMC PMC4643231
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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.

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  • Although 16S metabarcodes are extensively used for profiling modern microbiota, the approach is impracticable on ancient material because of amplification biases introduced during PCR due to extensive DNA fragmentation.

    “Although 16S metabarcodes [G] are extensively used for profiling modern microbiota, the approach is impracticable on ancient material because of amplification biases introduced during PCR due to extensive DNA fragmentation 133 .”

    From Ancient DNA analysis (2021) · cites this paper as [133]

    The Discussion says fragmented ancient DNA causes differential PCR amplification and taxonomic bias, limiting the method’s applicability to ancient microbiomes. But the Abstract says 16S amplicon sequencing may still be useful for qualitative screening, so calling it impracticable on ancient material without qualification overstates the paper’s conclusion. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Allelic drop out can occur due to clonal biases inherent in PCR or length polymorphisms in the target region, especially problematic when DNA is degraded and template copy number is low.

    “Second, allelic drop out, the failure of certain templates to amplify, can occur as a result of the clonal biases inherent in PCR or from length polymorphisms in the target region 133 , both of which are especially problematic when DNA is degraded and template copy number is low, giving rise to low or skewed sequence complexity.”

    From Ancient DNA analysis (2021) · cites this paper as [133]

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  • Ancient DNA analysis

    Orlando, L., Allaby, R., Skoglund, P., Der Sarkissian, C., Stockhammer, P. W., Ávila-Arcos, M. C., Fu, Q., Krause, J., Willerslev, E., Stone, A. C., et al. (2021) · Nature Reviews Methods Primers · doi:10.1038/s43586-020-00011-0 · Cited by 411

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Metadata

Authors
Ziesemer, K. A., Mann, A. E., Sankaranarayanan, K., Schroeder, H., Ozga, A. T., Brandt, B. W., Zaura, E., Waters-Rist, A., Hoogland, M., Salazar-García, D. C., Aldenderfer, M., Speller, C., Hendy, J., Weston, D. A., MacDonald, S. J., Thomas, G. H., Collins, M. J., Lewis, C. M., Hofman, C., & Warinner, C.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2015-11-13
Volume
5
Issue
1
ISSN
2045-2322
DOI
10.1038/srep16498
PMC
PMC4643231
Cited by
148
References
12
Language
en
Update notices
correction (2016-06-02)
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