Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array

Authors: Alison M. Devault, Kevin McLoughlin, Crystal Jaing, Shea Gardner, Teresita M. Porter, Jacob M. Enk, James Thissen, Jonathan Allen, Monica Borucki, Sharon N. DeWitte, Anna N. Dhody, and Hendrik N. Poinar

Scientific Reports, Vol. 4, Article No. 4245, March 2014, pp. 1–8; doi: 10.1038/srep04245


Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

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Ancient whole genome enrichment using baits built from modern DNA

Authors: Jacob Enk, Alison Devault, Melanie Kuch, Yusuf Murgha, Jean-Marie Rouillard, and Hendrik Poinar

Molecular Biology and Evolution, February 13, 2014. doi: 10.1093/molbev/msu074


We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of about 80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.

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Yersinia pestis and the Plague of Justinian 541—543 AD: a genomic analysis

Authors: David M Wagner PhD, Jennifer Klunk BS, Michaela Harbeck PhD, Alison Devault MA, Nicholas Waglechner MSc, Jason W Sahl PhD, Jacob Enk MSc, Dawn N Birdsell PhD, Melanie Kuch MSc, Candice Lumibao MSc, Debi Poinar MA, Talima Pearson PhD, Mathieu Fourment PhD, Prof Brian Golding PhD, Julia M Riehm PhD, Prof David J D Earn PhD, Sharon DeWitte PhD, Jean-Marie Rouillard PhD, Prof Gisela Grupe PhD, Ingrid Wiechmann PhD, Prof James B Bliska PhD, Prof Paul S Keim PhD, Holger C Scholz PhD, Prof Edward C Holmes PhD, Dr Hendrik Poinar PhD
The Lancet Infectious Diseases, Early Online Publication, 28 January 2014; doi:10.1016/S1473-3099(13)70323-2


Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14—17th centuries) and third (19—20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6—8th centuries). To address this gap, we sequenced and analysed draft genomes of Y. pestis obtained from two individuals who died in the first pandemic.
Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y. pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y. pestis strains, compared them with a database of genomes from 131 Y. pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree.
Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics.
We conclude that the Y. pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y. pestis into human populations.

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Second-Pandemic Strain of Vibrio cholerae from the Philadelphia Cholera Outbreak of 1849

Authors: Alison M. Devault, M.A., G. Brian Golding, Ph.D., Nicholas Waglechner, M.Sc., Jacob M. Enk, M.Sc., Melanie Kuch, M.Sc., Joseph H. Tien, Ph.D., Mang Shi, M.Phil., David N. Fisman, M.D., M.P.H., Anna N. Dhody, M.F.S., Stephen Forrest, M.Sc., Kirsten I. Bos, Ph.D., David J.D. Earn, Ph.D., Edward C. Holmes, Ph.D., and Hendrik N. Poinar, Ph.D.
New England Journal of Medicine 2014; 370:334-340 January 23, 2014 DOI: 10.1056/NEJMoa1308663

In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.

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