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Open Access Highly Accessed Research article

False positives complicate ancient pathogen identifications using high-throughput shotgun sequencing

Michael G Campana12*, Nelly Robles García3, Frank J Rühli12 and Noreen Tuross1

Author Affiliations

1 Department of Human Evolutionary Biology, Harvard University, Peabody Museum, 11 Divinity Avenue, Cambridge, MA 02138, USA

2 Centre for Evolutionary Medicine, Anatomy Institute, University of Zurich, 190 Winterthurerstrasse, Zurich 8057, Switzerland

3 Instituto Nacional de Antropología e Historia, Mexico City, Mexico

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BMC Research Notes 2014, 7:111  doi:10.1186/1756-0500-7-111

Published: 25 February 2014



Identification of historic pathogens is challenging since false positives and negatives are a serious risk. Environmental non-pathogenic contaminants are ubiquitous. Furthermore, public genetic databases contain limited information regarding these species. High-throughput sequencing may help reliably detect and identify historic pathogens.


We shotgun-sequenced 8 16th-century Mixtec individuals from the site of Teposcolula Yucundaa (Oaxaca, Mexico) who are reported to have died from the huey cocoliztli (‘Great Pestilence’ in Nahautl), an unknown disease that decimated native Mexican populations during the Spanish colonial period, in order to identify the pathogen. Comparison of these sequences with those deriving from the surrounding soil and from 4 precontact individuals from the site found a wide variety of contaminant organisms that confounded analyses. Without the comparative sequence data from the precontact individuals and soil, false positives for Yersinia pestis and rickettsiosis could have been reported.


False positives and negatives remain problematic in ancient DNA analyses despite the application of high-throughput sequencing. Our results suggest that several studies claiming the discovery of ancient pathogens may need further verification. Additionally, true single molecule sequencing’s short read lengths, inability to sequence through DNA lesions, and limited ancient-DNA-specific technical development hinder its application to palaeopathology.

True single molecule sequencing; High-throughput sequencing; Pathogen; Ancient DNA; False positive