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A Tale of 3 Mummies: A Microbiome Analysis of Life in the Peruvian Andes 1,000 Years Ago

Apr 14, 2016 at 12:00 PM ET

Abstract

The natural mummification process is a rare and unique process resulting from low temperatures and oxygen levels, and dry weather conditions. In the present study, we characterized the gut microbiome of three pre-Columbian Andean mummies using 16S rRNA gene high-throughput sequencing and metagenomics to understand the preservation and evolution of commensal and pathogenic microorganisms, antimicrobial resistance genes, diet, and the metabolic processes during the natural mummification of the human gut.

Key Points

  • 16S rRNA gene high-throughput sequencing and metagenomics can be used to understand changes to the human microbiome
  • Evaluation of microbial populations from mummified remains can provide valuable insight into the medical and cultural aspects of ancient people
  • Ancient DNA must be suitable for investigation and should be evaluated through damage analysis studies
  • Quality control of the metagenome analysis process is key to ALL studies. It is particularly important in ancient DNA studies to ensure that the results are reflective of the actual sample, not an artifact of DNA extraction, PCR, sequencing, and/or analysis

Raul Cano, headshot.

Raul Cano, PhD

Director, Microbiome Research, ATCC-CTM and Professor Emeritus, California Polytechnic State University

Raul Cano, Ph.D., is a Professor Emeritus at California Polytechnic State University and the Director, Microbiome Research at the ATCC Center for Translational Microbiology (ATCC-CTM). He has extensive experience in the fields of microbial ecology, bioremediation, metagenomics, clinical microbiology, and microbiome research. Dr. Cano was named a Fellow of the American Academy of Microbiology and has received several awards for his outstanding teaching skills, including the 1997 Carski Foundation Distinguished Teacher Award.

Questions and Answers

Did you do the binning analyses with the reads or the assembled reads (contigs)?

We did the binning analysis using the individual reads.

Do you have any plans to study mummies of other origins, ages, and cultures? And, are there likely artifacts associated with specific mummification processes?

Yes, we do. We are currently waiting for results from two sets of natural mummies, one set from Libya and another from an abbey in Northern Italy. We are also looking at preColombian samples from the Caribbean.

Does the affordability of a meat-based diet for Italian nobility versus the Inca population have any bearing on the differences you see?

Probably not. Rather, it is likely that cultural habits and agricultural practices played a role in the differences we saw.

How did you ascertain that the sequences you are analyzing originated from the mummy and not the environment?

We used multiple approaches starting with aseptic surgical procedures in a surgical suite to take the biopsy samples. We also used a variety of bioinformatics tools, including mapDamage, Source Tracker, and Operational Taxonomic Unit (OUT) subtraction from control DNA extractions.

How did you determine the diet of the Pre-Columbian European mummies? Is it based on the number of hits to a particular database?

We interrogated the NCBI databases with individual reads using the blastx algorithm. After the results were obtained, they were sorted based on e value, % identity, sequence length, and best hit. The number of hits as well as the relative abundance values were used to identify the specific plant and animal genes reported.

Why did you think the alpha diversity of the Pre-Columbian mummies was lower compared to the Italian mummies?

It is likely that the relatively restricted vegetarian diet of the Pre-Columbian mummies “selected” for a less diverse, more specialized, carbohydrate-digesting microbiota.

In the network analysis of the OTUs, why did you think there are three different clusters for the Pre-Columbian mummies and just one big cluster for the Italian mummies? Does it have to do with diet, culture, and also possibly disease?

This is likely as the Italian nobility mummies had a more homogeneous, carnivorous diet as well as similar cultural habits. In contrast, the Peruvian mummies represented three different groups; one from the pre-Inca period (likely prior to the broad-based use of quinoa as a protein source), and the other two representing a male Inca who probably traveled more extensively than the female Inca, with more restricted travel, which led to different dietary compositions of their corresponding diet.

Would you please explain how the control bacteria (10 to 16 of them as shown in one of your slides) were used to ensure the DNA purification and extraction efficiencies? Were they spiked into the experimental sample? Also, why were these strains chosen?

The mock community consisted of 11 species of bacteria representing the spectrum of cell wall type, G+C ratios, oxygen requirements, and genome size. They were mixed in “approximately” equal densities of 1 x 106 CFUs and ran as an additional sample, in parallel, during DNA extraction, library construction, NGS, and QIIME analysis. They were not spiked as they would have obfuscated the results.