Shotgun microbial profiling of fossil remains – University of Copenhagen

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24 March 2014

Shotgun microbial profiling of fossil remains

In a study entitled “Shotgun Microbial Profiling of Fossil Remains” and published in Molecular Ecology, researchers from the Orlando group describe the diversity of microbes that have colonized the remains of ca. 200-13,000 years old horses.

With this work, the authors demonstrate that the amount of genetic information that can be retrieved from fossils can be increased significantly.

Ancient genomics: sequencing genomes of long-dead individuals and more

The field of ancient DNA has undergone a recent revolution with developments in ultra-high throughput sequencing technologies. These have made possible the characterization of complete nuclear genomes from long-dead organisms, such as the 4,000-year-old Saqqaq Eskimo and the 700,000-year-old horse sequenced at the Centre for Geogenetics.

The reconstruction of these ancient genomes relies on shotgun sequencing, a technique that recovers genetic information from the entire population of DNA molecules extracted from a sample. Shotgun sequencing rapidly revealed that an ancient DNA extract is in fact a mixture of DNA molecules of various origins, including some derived from the specimen of interest (endogenous DNA), while the rest arises from environmental microbes feeding on the fossil material.

The percentage of endogenous DNA can exceptionally be as high 70% but typically represent no more than a few percents. This mainly results from a variety of diagenetic mechanisms that fragment and degrade the DNA molecules preserved in fossils. A practical consequence of this is that high-throughput shotgun sequencing often produces a vast majority of the genetic sequences that are not relevant to the genetic study of the specimen.

Microbial diversity of ancient horse remains from the Siberian permafrost

With this study, the authors find a new way to make use of this information data that is routinely disregarded by exploiting the molecular signatures of microbes. Der Sarkissian et al. utilize a suite of computational methods for microbial profiling and ecological analyses to shotgun datasets obtained from seven ca. 200-13,000 years old horses.

These horses were collected in the permafrost of Yakutia and the Taymir Peninsula in Siberia, in conditions considered favorable for ancient DNA preservation. Even in these optimal conditions, the endogenous horse DNA content could be as low as 0.4%!

Using taxonomy-based assignment methods capable of identifying bacterial taxa and calculating their relative abundances in a robust manner, the main bacterial genera identified were Mycobacterium, Pseudomonas and Rhodopseudomonas, and as a whole, the microbial diversity observed in the ancient horse samples was found to be typical of soil samples. Microbial differences among samples are thought to reflect differences in the depositional micro-environment of the samples.

Preservation niches

Another result of this study is that the same microbes were found both in the deepest and most superficial parts of the horse bones and teeth. This is not consistent with the hypothesis according to which particular groups of microbes would be responsible for the more extensive degradation of ancient DNA molecules that is observed when extracting them from the sample´s exterior rather than from its interior. Conversely, the results of this study support the existence of preservation niches in crystal aggregates in the most interior parts of ancient samples, where endogenous DNA molecules are found in higher amounts and in a less degraded state.

Making the most of ancient shotgun sequencing data

In addition to increase the amount of information that can be gathered from the diversity of molecules retrieved from ancient fossils, the framework applied in this study can help better understanding the “post-mortem life” of a sample. These methods could indeed provide interesting insights into the different microbial signatures of ancient samples and there relationships to types of samples, depositional environments or post-collection storage conditions, if this framework is applied to a wider range of shotgun datasets generated from ancient samples varying in age and geographical origin.

This knowledge could later be used to design novel extraction and analysis methods tailored to ancient endogenous DNA. Another prospect for the analyses presented in this article could be the study of host-associated microbiomes and pathogenicity.

The article was published online in Molecular Ecology: https://onlinelibrary.wiley.com/doi/10.1111/mec.12690/abstract

Reference: C. Der Sarkissian, L. Ermini, H. Jónsson, A. N. Alekseev, E. Crubezy, B. Shapiro, L. Orlando 2014, ' Shotgun Microbial Profiling of Fossil Remains. ' Molecular Ecology.