Comparison of extraction methods for recovering ancient microbial DNA from paleofeces

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces

Richard W. Hagan, Courtney A. Hofman, Alexander Hübner, Karl Reinhard, Stephanie Schnorr, Cecil M. Lewis Jr, Krithivasan Sankaranarayanan, Christina G. Warinner

Paleofeces, or coprolites, can provide insight into diet, ecology, and nutritional information. Currently, there is no standard DNA extraction method. This study used shotgun metagenomics to compare five DNA extraction methods on both human and dog paleofeces (~1,800 BP). Paleofeces is difficult to work with as it is degraded, like most ancient DNA, and the extraction method can impact the microbial reconstruction.

Standard methods for fresh feces and ancient feces vary due to the nature of aDNA and extraction process. Unfortunately, this doesn’t provide a clear picture to compare the two data sets. It is important to create a better set of standards that can be applied to both samples in order to yield optimal results while creating less bias.

The five protocols used were: “…Method A: Human Microbiome Project (HMP) Protocol with PowerSoil Kit; Method B: Modified MinElute Protocol; Method C: Phenol-Chloroform + Modified MinElute Protocol; Method D: Split Modified MinElute Protocol; Method E: HMP Protocol + Modified MinElute Protocol (Hagan et al., 2020).”

The lowest DNA yield following these methods were Methods A and E, with Method A also providing the lowest DNA recovery. Methods B, C, and D performed well with minimal differences. Method D provided the highest DNA yield (median) and was less complicated than Method B. Method C uses chloroform purification, therefore this study did not recommend this method due to the harsh chemicals used and not performing any better than the other methods. Method D was recommended, due to the ease of use and DNA recovery, as well as Method B.

While the DNA extraction methods all provided a microbial taxonomic profile, the ancient methods provided more DNA to work with. The aDNA methods did not negatively impact the reconstructed microbial communities, with all DNA extractions test yielding similar communities.

Overall, when conducting future comparative studies in paleofeces (aDNA and contemporary), using Methods B and D are recommended, but favoring D if issues occur during spin filtration.

Kailie Batsche

AJPA update (February 2020)

Link: https://onlinelibrary.wiley.com/doi/full/10.1002/ajpa.23978

Hagan, R. W., Hofman, C. A., Hübner, A., Reinhard, K., Schnorr, S., Lewis, C. M., Sankaranarayanan, K., & Warinner, C. G. (2020). Comparison of extraction methods for recovering ancient microbial DNA from paleofeces. American Journal of Physical Anthropology, 171(2), 275–284. https://doi.org/10.1002/ajpa.23978