Abstract
Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods, such as restriction‐site‐associated
...DNA
sequencing (
RAD
seq) and sequence capture, are constrained by costs associated with inefficient use of sequencing data and sample preparation. Here, we introduce
RAD
cap, an approach that combines the major benefits of
RAD
seq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches.
RAD
cap uses a new version of dual‐digest
RAD
seq (3
RAD
) to identify candidate
SNP
loci for capture bait design and subsequently uses custom sequence capture baits to consistently enrich candidate
SNP
loci across many individuals. We combined this approach with a new library preparation method for identifying and removing
PCR
duplicates from 3
RAD
libraries, which allows researchers to process
RAD
seq data using traditional pipelines, and we tested the
RAD
cap method by genotyping sets of 96–384
Wisteria
plants. Our results demonstrate that our
RAD
cap method: (i) methodologically reduces (to <5%) and allows computational removal of
PCR
duplicate reads from data, (ii) achieves 80–90% reads on target in 11 of 12 enrichments, (iii) returns consistent coverage (≥4×) across >90% of individuals at up to 99.8% of the targeted loci, (iv) produces consistently high occupancy matrices of genotypes across hundreds of individuals and (v) costs significantly less than current approaches.
Gilbert et al. (Reports, 9 May 2008, p. 786) presented DNA analysis of coprolites recovered from an Oregon cave as evidence for a human presence in North America before the Clovis culture. Results of ...our micromorphological and Fourier transform infrared spectroscopy analyses of one of the reported coprolites are difficult to reconcile with the DNA results identifying the coprolite as human.
Pregnancy complications are poorly represented in the archeological record, despite their importance in contemporary and ancient societies. While we excavated a Byzantine cemetery in Troy, we ...discovered calcified abscesses among a woman’s remains. Scanning electron microscopy of the tissue revealed ‘ghost cells’, resulting from dystrophic calcification, which preserved ancient maternal, fetal and bacterial DNA of a severe infection, likely chorioamnionitis.Gardnerella vaginalisandStaphylococcus saprophyticusdominated the abscesses. Phylogenomic analyses of ancient, historical, and contemporary data showed thatG. vaginalisTroy fell within contemporary genetic diversity, whereasS. saprophyticusTroy belongs to a lineage that does not appear to be commonly associated with human disease today. We speculate that the ecology ofS. saprophyticusinfection may have differed in the ancient world as a result of close contacts between humans and domesticated animals. Our results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections.
After evolving in Africa at the close of the Miocene, mammoths (Mammuthus sp.) spread through much of the northern hemisphere, diversifying morphologically as they entered various habitats. ...Paleontologically, these morphs are conventionally recognized as species. In Pleistocene North America alone, several mammoth species have been recognized, inhabiting environments as different as cold tundra-steppe in the north and the arid grasslands or temperate savanna-parklands of the south. Yet mammoth phylogeographic studies have overwhelmingly focused on permafrost-preserved remains of only one of these species, Mammuthus primigenius (woolly mammoth). Here we challenge this bias by performing a geographically and taxonomically wide survey of mammoth genetic diversity across North America. Using a targeted enrichment technique, we sequenced 67 complete mitochondrial genomes from non-primigenius specimens representing M. columbi (Columbian mammoth), M. jeffersonii (Jeffersonian mammoth), and M. exilis (pygmy mammoth), including specimens from contexts not generally associated with good DNA preservation. While we uncovered clear phylogeographic structure in mammoth matrilines, their phylogeny as recovered from mitochondrial DNA is not compatible with existing systematic interpretations of their paleontological record. Instead, our results strongly suggest that various nominal mammoth species interbred, perhaps extensively. We hypothesize that at least two distinct stages of interbreeding between conventional paleontological species are likely responsible for this pattern – one between Siberian woolly mammoths and resident American populations that introduced woolly mammoth phenotypes to the continent, and another between ecomorphologically distinct populations of woolly and Columbian mammoths in North America south of the ice.
Genomic data contribute invaluable information to the epidemiological investigation of pathogens of public health importance. However, whole-genome sequencing (WGS) of bacteria typically relies on ...culture, which represents a major hurdle for generating such data for a wide range of species for which culture is challenging. In this study, we assessed the use of culture-free target-enrichment sequencing as a method for generating genomic data for two bacterial species: (1)
which causes anthrax in both people and animals and whose culture requires high-level containment facilities; and (2)
, a fastidious emerging human respiratory pathogen. We obtained high-quality genomic data for both species directly from clinical samples, with sufficient coverage (>15×) for confident variant calling over at least 80% of the baited genomes for over two thirds of the samples tested. Higher qPCR cycle threshold (
) values (indicative of lower pathogen concentrations in the samples), pooling libraries prior to capture, and lower captured library concentration were all statistically associated with lower capture efficiency. The
value had the highest predictive value, explaining 52 % of the variation in capture efficiency. Samples with
values ≤30 were over six times more likely to achieve the threshold coverage than those with a
> 30. We conclude that target-enrichment sequencing provides a valuable alternative to standard WGS following bacterial culture and creates opportunities for an improved understanding of the epidemiology and evolution of many clinically important pathogens for which culture is challenging.
Yersinia pestishas 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 ofY pestisobtained from two individuals who died in the first pandemic. Methods 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 theY pestis-specificplagene 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 infectiousY pestisstrains, compared them with a database of genomes from 131Y pestisstrains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. Findings 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. Interpretation We conclude that theY pestislineages 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 ofY pestisinto human populations. Funding McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.
Smallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there ...have been claims of smallpox in Egypt, India, and China dating back millennia 1-4, the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial 4-9. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries 4-6 and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics 1, 2, 10. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20
century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18
and 19
centuries, concomitant with the development of modern vaccination.
Palaeogenetic research on human pathogenic and microbiomic bacteria has been largely restricted to bloodborne pathogens from skeletal tissue and, due to short lengths of degraded ancient DNA, ...small-scale single loci studies. My thesis has expanded the breadth and depth of palaeomicrobial knowledge via the study of novel specimen types with next-generation technologies. Presented in sandwich thesis format, I discuss genome-scale studies of three previously-unstudied historical pathogens: 19th century Vibrio cholerae (cholera) from an alcohol-preserved intestine from Philadelphia, and medieval Staphylococcus saprophyticus (urinary tract infections) and Gardnerella vaginalis (bacterial vaginosis) from calcified urogenital infections of a Trojan woman. Cholera persists as a dangerous modern disease that was also responsible for severe historic epidemics. My research confirms that 19th century pandemics were caused by an O1 classical strain that may have possessed genomic features that contributed increased virulence. S. saprophyticus and G. vaginalis are opportunistic pathogens of the urogenital microbiome, especially in reproductive-age females. Using very high endogenous DNA content of the calcified infections, I have reconstructed one of the most complete ancient bacterial genomes for S. saprophyticus and coding genome for G. vaginalis. Both ancient pathogens possess most of the virulence and urogenital adaptive genes of modern strains, indicating similar ecological roles for these species in past female health. Finally, I successfully use LLMDA microarray technology (never before utilized for ancient DNA research) to detect ancient pathogens. LLMDA provides an inexpensive and informative alternative to high-throughput sequencing for assessing the metagenomic content of ancient samples. Together, my findings provide a framework emphasizing the need to broadly study past microbiomes in conjunction with specific pathogens. Using molecular data, this work supports anthropological views of infectious disease ecology related to the first epidemiological transition and historical narratives. Taken together with the recent literature on ancient pathogen genomes, my findings indicate that palaeogenome sequences may not necessarily reveal any specific signatures of greater virulence, and interpretations of past diseases must necessarily take into account additional host, environmental, and cultural factors.
Thesis
Doctor of Philosophy (PhD)
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