Comparison of two ancient DNA extraction protocols for skeletal remains from tropical environments

Objectives The tropics harbor a large part of the world\u27s biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semi‐tropical sites in Tanzania, Mexico, and Puerto Rico (N = 12). Materials and Methods All samples were extracted twice, built into double‐stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step. Results No significant difference was found in overall ancient DNA yields or post‐mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality‐filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments. Discussion Both methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future

Chimpanzees of the Past: Full Mitochondrial Genomes from Pan Troglodytes schweinfurthii Skeletons from Gombe National Park

The tropical environment of Gombe National Park, Tanzania makes the recovery of endogenous genetic material from skeletal remains inherently difficult. Since the 1960’s many of the Pan troglodytes schweinfurthii skeletons were interred under circumstances not conducive to adequate DNA preservation. To date, no skeletal material has yielded usable DNA, despite the relatively young age of the samples. Here, for the first time, using novel extraction methods, we capture chimpanzee DNA from dentin and calcified dental plaque (calculus) in order to address questions about overall preservation and genetic diversity within several generations of Gombe National Park inhabitants. DNA was extracted from a total of 28 chimpanzees (9 dentin and 28 calculus samples) and built into double-stranded shotgun libraries. Amplified libraries underwent in-solution hybridization capture enrichment and were sequenced on an Illumina MiSeq (v2, 2x150 paired end) in order to obtain complete mitochondrial genomes (mitogenomes). We were able to reconstruct successfully the mitogenomes from all 9 dentin samples (240x mean coverage at 100%) and 28 calculus samples (37x mean coverage at 98%). We used mitogenomes to conduct an exploratory survey of genetic diversity over time at Gombe. When combined with publicly available P.t. schweinfurthii HV1 data, we found that haplotype diversity of chimpanzees who died before 1992 was slightly higher than those who died after (0.862 compared to 0.824). These preliminary results mark the first mitogenomes reconstructed from deceased Gombe chimpanzee calculus and further validate the use of the biological material as a reservoir for host DNA

Comparison of aDNA Yields from Calculus and Tooth Roots in Pre-Columbian Skeletal Remains

In recent years, dental calculus has emerged as an important source of ancient genetic material. However, calculus has not been extensively utilized as a source of endogenous host DNA when working with human skeletal remains. In this study we compare endogenous DNA yields obtained from extractions performed from both dental calculus and dental tooth roots for three pre-Columbian individuals, originating from three different archaeological sites of the island of Puerto Rico. Furthermore, in order to assess the effects of physical decontamination procedures on recovery of endogenous DNA, tooth root samples were further subdivided into two groups: one group was treated by removal of the cementum and the second group was left untreated. Extractions were then performed in three replicates for each individual, one from calculus, one from treated tooth roots and one from untreated tooth roots. DNA extracts were quantified, transformed into sequencing libraries, and enriched for the complete mitochondrial genome through in-solution hybridization capture. Preliminary results indicate that out of nine extracts obtained (three replicates for each individual) only seven were successfully built into libraries. Sequence data suggest that libraries made from treated tooth root extracts contain, on average, more sequence reads mapping to the reference and higher coverage than libraries built from untreated tooth root or calculus extracts. These results suggest that although calculus is a viable source of endogenous DNA, treated tooth root extractions result in higher overall endogenous DNA yields and a reduced presence of contaminant DNA molecules in these samples

Ancient Dental Calculus as a Reservoir of Whole Human Mitogenomes

Until recently, one challenge of ancient DNA research has been the necessary destruction of skeletal material in order to extract human genetic information. Calcified dental plaque (dental calculus) can be removed from dentition without damaging the underlying teeth or surrounding bone and it has been shown to be an excellent reservoir of microbial and dietary biomolecules and microfossils. Here, we examine the preservation of human mitochondrial DNA in dental calculus by performing high-throughput mitogenome sequencing of material from six individuals from Norris Farms #36, a Mississippian period Oneota cemetery in Illinois dating back to 700BP. DNA was extracted in a dedicated ancient DNA facility at the University of Oklahoma and built into shotgun libraries. Following library amplification, each sample underwent a mitochondrial DNA capture and enrichment protocol at Arizona State University and was sequenced on an Illumina MiSeq platform. Medium coverage (7-34x) whole mitogenomes were successfully recovered from all six samples from Norris Farms #36 including three which were previously unsuccessful for HVRI sequencing using traditional PCR-based methods. In one calculus sample, the proportion of endogenous mtDNA increased from \u3c0.0001% in unenriched shotgun to 0.34% using mitochondrial capture and enrichment. This research demonstrates that calculus, an abundant and ubiquitous archaeological substrate, contains sufficient host information for whole mitogenome reconstruction. These findings have important implications for the use of dental calculus as an alternative material in archaeological studies of maternal ancestry

Paleogenomic insights into the red complex bacteria <i>Tannerella forsythia</i> in Pre-Hispanic and Colonial individuals from Mexico

The ‘red complex’ is an aggregate of three oral bacteria (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) responsible for severe clinical manifestation of periodontal disease. Here, we report the first direct evidence of ancient T. forsythia DNA in dentin and dental calculus samples from archaeological skeletal remains that span from the Pre-Hispanic to the Colonial period in Mexico. We recovered twelve partial ancient T. forsythia genomes and observed a distinct phylogenetic placement of samples, suggesting that the strains present in Pre-Hispanic individuals likely arrived with the first human migrations to the Americas and that new strains were introduced with the arrival of European and African populations in the sixteenth century. We also identified instances of the differential presence of genes between periods in the T. forsythia ancient genomes, with certain genes present in Pre-Hispanic individuals and absent in Colonial individuals, and vice versa. This study highlights the potential for studying ancient T. forsythia genomes to unveil past social interactions through analysis of disease transmission. Our results illustrate the long-standing relationship between this oral pathogen and its human host, while also unveiling key evidence to understand its evolutionary history in Pre-Hispanic and Colonial Mexico. This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules'

Genomic insights into the early peopling of the Caribbean

The Caribbean was one of the last regions of the Americas to be settled by humans, but where they came from and how and when they reached the islands remain unclear. We generated genome-wide data for 93 ancient Caribbean islanders dating between 3200 and 400 calibrated years before the present and found evidence of at least three separate dispersals into the region, including two early dispersals into the Western Caribbean, one of which seems connected to radiation events in North America. This was followed by a later expansion from South America. We also detected genetic differences between the early settlers and the newcomers from South America, with almost no evidence of admixture. Our results add to our understanding of the initial peopling of the Caribbean and the movements of Archaic Age peoples in the Americas.The research was funded by the Max Planck Society and the European Research Council under the 7th Framework Program (grant agreement no. 319209, ERC Synergy Project NEXUS1492). H.S. was supported by the HERA (Humanities in the European Research Area) Joint Research Program “Uses of the Past” (CitiGen) and the European Union’s Horizon 2020 research and innovation program under grant agreement no. 649307. W.J.P. and M.A.N.-C. were supported by the National Science Foundation (BCS-0612727 and BCS1622479). C.L.-F. was supported by a grant from the Ministry of Science, Innovation and Universities (PGC2018-0955931-B-100, AEI/FEDER, UE). M.R. was supported by the Social Sciences and Humanities Research Council of Canada (435-2016-0529). M.R., Y.C.d.A., U.M.G.H., and S.T.H.G. were supported by the Social Sciences and Humanities Research Council of Canada (standard research grant SSHRC ‐ 410‐2011‐1179 and SSHRC postdoctoral fellowship ‐ 756‐2016‐0180) and several University of Winnipeg internal grants (Major grant 2017, 2018; Partnership Development grant 2017, 2018; and Discretionary grant 2017, 2018)

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field