Abstract
BackgroundIdentification 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.ResultsWe 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.ConclusionsFalse 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.
Highlights
Identification of historic pathogens is challenging since false positives and negatives are a serious risk
Environmental contamination is a critical issue in ancient Deoxyribonucleic acid (DNA) investigations of diseases
Previous investigations of ancient pathogens using both polymerase chain reaction (PCR) and highthroughput sequencing may need to be re-evaluated because pathogen identifications require extensive verification procedures due to the high risk of false positives
Summary
Identification of historic pathogens is challenging since false positives and negatives are a serious risk. High-throughput sequencing may help reliably detect and identify historic pathogens. The pathogens responsible for many historic epidemics remain either unknown or speculative (e.g. the Plague of Athens [1,2]). Identification of these historic pathogens is critical to understanding disease evolution, which in turn has direct impacts on the development of effective medical treatments for these conditions. Due to the limitations of molecular preservation and detection, negative assays do not constitute evidence of absence of any infection [15,16,17]. Many PCR-based palaeopathological reports are contentious due to insufficient anti-contamination procedures or lack of experimental and analytical rigor [21,22,23]
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