Objectives Archaeological dental calculus is a rich source of host-associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Materials and Methods Extracted DNA from six individuals at the 700-year-old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in-solution capture techniques, followed by Illumina high-throughput sequencing. Results Full mitogenomes (7-34x) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Discussion Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains.

Leprosy is caused by the bacterial pathogens Mycobacterium leprae and Mycobacterium lepromatosis. Apart from humans, animals such as nine-banded armadillos in the Americas and red squirrels in the British Isles are naturally infected with M. leprae. Natural leprosy has also been reported in certain nonhuman primates, but it is not known whether these occurrences are due to incidental infections by human M. leprae strains or by M. leprae strains specific to nonhuman primates. In this study, complete M. leprae genomes from three naturally infected nonhuman primates (a chimpanzee from Sierra Leone, a sooty mangabey from West Africa, and a cynomolgus macaque from The Philippines) were sequenced. Phylogenetic analyses showed that the cynomolgus macaque M. leprae strain is most closely related to a human M. leprae strain from New Caledonia, whereas the chimpanzee and sooty mangabey M. leprae strains belong to a human M. leprae lineage commonly found in West Africa. Additionally, samples from ring-tailed lemurs from the Bezà Mahafaly Special Reserve, Madagascar, and chimpanzees from Ngogo, Kibale National Park, Uganda, were screened using quantitative PCR assays, to assess the prevalence of M. leprae in wild nonhuman primates. However, these samples did not show evidence of M. leprae infection. Overall, this study adds genomic data for nonhuman primate M. leprae strains to the existing M. leprae literature and finds that this pathogen can be transmitted from humans to nonhuman primates as well as between nonhuman primate species. While the prevalence of natural leprosy in nonhuman primates is likely low, nevertheless, future studies should continue to explore the prevalence of leprosy-causing pathogens in the wild. Author summaryMycobacterium leprae, which causes leprosy in humans, also infects nine-banded armadillos, red squirrels, and nonhuman primates. Genomic data for M. leprae strains from wild armadillos and red squirrels show that humans were responsible for the original introduction of M. leprae to these species. It is not known whether naturally occurring leprosy among nonhuman primates is due to incidental infections from humans or whether nonhuman primates can serve as a host for M. leprae. To this end, we sequenced complete genomes of M. leprae strains from three naturally infected nonhuman primates. Our results suggest that M. leprae strains can be transmitted from humans to nonhuman primates as well as between nonhuman primate species, and thus, other primates might serve as a host for M. leprae in the wild. We also assessed whether wild ring-tailed lemurs from Madagascar and chimpanzees from Uganda showed presence of M. leprae infection. Although these populations tested negative for M. leprae infection, further research on the prevalence of M. leprae in other wild nonhuman primate populations, especially in leprosy-endemic regions, is warranted.

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CrAssphage (cross-assembly phage) is a bacteriophage that was first discovered in human gut metagenomic data. CrAssphage belongs to a diverse family of crAss-like bacteriophages thought to infect gut commensal bacteria belonging to Bacteroides species. However, not much is known about the biogeography of crAssphage and whether certain strains are associated with specific human populations. In this study, we screened publicly available human gut metagenomic data from 3,341 samples for the presence of crAssphage sensu stricto (NC_024711.1). We found that crAssphage prevalence is low in traditional, hunter-gatherer populations, such as the Hadza from Tanzania and Matses from Peru, as compared to industrialized, urban populations. Statistical comparisons showed no association of crAssphage prevalence with variables such as age, sex, body mass index, and health status of individuals. Phylogenetic analyses show that crAssphage strains reconstructed from the same individual over multiple time-points, cluster together. CrAssphage strains from individuals from the same study population do not always cluster together. Some evidence of clustering is seen at the level of broadly defined geographic regions, however, the relative positions of these clusters within the crAssphage phylogeny are not well-supported. We hypothesize that this lack of strong biogeographic structuring is suggestive of an expansion event within crAssphage. Using a Bayesian dating approach, we estimate that this expansion has occurred fairly recently. Overall, we determine that crAssphage presence is associated with an industrialized lifestyle and the absence of strong biogeographic structuring within global crAssphage strains is likely due to a recent population expansion within this bacteriophage.

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

Many studies investigating the human microbiome-cancer interface have focused on the gut microbiome and gastrointestinal cancers. Outside of human papillomavirus driving cervical cancer, little is known about the relationship between the vaginal microbiome and other gynecological cancers, such as ovarian cancer. In this retrospective study, we investigated the relationship between ovarian cancer, platinum-free interval (PFI) length, and vaginal and gut microbiomes. We observed that Lactobacillus-dominated vaginal communities were less common in women with ovarian cancer, as compared to existing datasets of similarly aged women without cancer. Primary platinum-resistance (PPR) disease is strongly associated with survivability under one year, and we found over one-third of patients with PPR (PFI < 6 months, n = 17) to have a vaginal microbiome dominated by Escherichia (>20% relative abundance), while only one platinum super-sensitive (PFI > 24 months, n = 23) patient had an Escherichia-dominated microbiome. Additionally, L. iners was associated with little, or no, gross residual disease, while other Lactobacillus species were dominant in women with >1 cm gross residual disease. In the gut microbiome, we found patients with PPR disease to have lower phylogenetic diversity than platinum-sensitive patients. The trends we observe in women with ovarian cancer and PPR disease, such as the absence of Lactobacillus and presence of Escherichia in the vaginal microbiome as well as low gut microbiome phylogenetic diversity have all been linked to other diseases and/or pro-inflammatory states, including bacterial vaginosis and autoimmune disorders. Future prospective studies are necessary to explore the translational potential and underlying mechanisms driving these associations.

31CrAssphage (cross-assembly phage) is a bacteriophage that was first discovered in 32 human gut metagenomic data. CrAssphage belongs to a diverse family of crAss-like 33 bacteriophages thought to infect gut commensal bacteria belonging to Bacteroides species. 34However, not much is known about the biogeography of crAssphage and whether certain strains 35 are associated with specific human populations. In this study, we screened publicly available 36 human gut metagenomic data from 3,341 samples for the presence of crAssphage sensu stricto 37 (NC_024711.1). We found that crAssphage prevalence is low in traditional, hunter-gatherer 38 populations, such as the Hadza from Tanzania and Matses from Peru, as compared to 39 industrialized, urban populations. Statistical comparisons showed no association of crAssphage 40 prevalence with variables such as age, sex, body mass index, and health status of individuals. 41Phylogenetic analyses show that crAssphage strains reconstructed from the same individual over 42 multiple time-points, cluster together. CrAssphage strains from individuals from the same study 43 population do not always cluster together. Some evidence of clustering is seen at the level of 44 broadly defined geographic regions, however, the relative positions of these clusters within the 45 crAssphage phylogeny are not well-supported. We hypothesize that this lack of strong 46 biogeographic structuring is suggestive of a recent expansion event within crAssphage. Using a 47Bayesian dating approach, we estimate this expansion has occurred within the past 200 years. 48Overall, we determine that crAssphage presence is associated with an industrialized lifestyle. 49The absence of strong biogeographic structuring within global crAssphage strains is likely due to 50 a recent population expansion within this bacteriophage. 51 52