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Contamination controls when preparing archaeological remains for ancient DNA analysis

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Abstract

Contamination is of utmost concern when working with ancient DNA as it easily leads to false positive results. The best way to prevent or minimize contamination is to start precautionary measures as early as possible, ideally commencing with sample collection and preparation by field archaeologists. This paper discusses the nature of contamination in ancient DNA studies and offers some practical guidelines as to how archaeologists in the field can “clean-collect” samples for ancient DNA analysis. Methods for preparing contaminated samples from museum collections for ancient DNA analysis are also discussed.

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... The analyzed specimens originated from various excavation contexts and were all morphologically identified as houndsharks (see Supplementary Table 1 for details). Decontamination, DNA extraction, and PCR setup procedures were performed in a positively pressured laboratory with a UV-HEPA air filtration system in the Department of Archaeology, Simon Fraser University (Burnaby, BC, Canada), that is dedicated to the analysis of aDNA (Cooper & Poinar, 2000;Yang & Watt, 2005). Following Speller and colleagues (2012), all the specimens were decontaminated prior to DNA extraction using a combination of bleach and UV light. ...
... All PCR amplifications and procedures involving amplified DNA were carried out in a negatively pressured post-PCR laboratory located in a different building than the aDNA laboratory (Cooper & Poinar, 2000;Yang & Watt, 2005). PCR amplifications were performed on a Mastercycler Personal or Gradient thermal cycler (Eppendorf) in a 30 µL reaction volume that included 1.5 × PCR Gold Buffer (Applied Biosystems), 2 mM MgCl 2 , 0.2 mM of each Fig. 3 Four of the shark vertebrae specimens (SHK18, SHK19, SHK20, and SHK21) from Point Alones that were analyzed in this study dNTP, 0.3 μM of primer Shark COI-MINIR, 3 μM of primer FishF1or FishF2, 1 mg/mL BSA, 3 μl DNA sample, and 0.75-1 U AmpliTaq Gold (Applied Biosystems). ...
... Upon an organism's death, the mechanisms that repaired its DNA while it was living cease, exposing their DNA to degradative processes that reduce the quantity and quality of DNA preserved within their remains (Dabney et al., 2013). As a result of this degradation, archaeological materials are susceptible to contamination with modern DNA and carryover contamination from previous amplifications (Cooper & Poinar, 2000;Yang & Watt, 2005). While the occurrence of contamination can never be entirely ruled out, the balance of probabilities suggest our a DNA results are authentic. ...
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Prior to burning down in 1906 CE, Point Alones in the Monterey Bay region of Central California was home to one of the largest Chinese fishing communities in the United States of America. Both historical records and the recovery of numerous cartilaginous fish (Chondrichthyes) vertebrae during archaeological excavations of the village indicate sharks were among the taxonomic groups being regularly harvested by its inhabitants. However, as shark vertebrae are difficult to identify past the family-level using conventional morphology-based approaches, our understanding of the Point Alones shark fishery remains incomplete. In this study, we address this issue by using ancient DNA analysis to assign species-level identifications to a sample of 54 shark vertebrae from the site. We successfully amplified a 173 bp fragment of the mitochondrial cytochrome c oxidase I gene from 47 of the 54 analyzed specimens (87.03%). Our results indicate that Tope Shark (Galeorhinus galeus; n = 39) was the primary focus of the site’s shark fishery, with Brown Smooth-Hound (Mustelus henlei; n = 7) and Leopard Shark (Triakis semifasciata; n = 1) also harvested to a lesser extent. All three of these species are found locally in the waters overlying the continental shelf, suggesting Chinese fishers were harvesting sharks from these coastal environments. While some of the sharks caught by fishers from Point Alones were likely being consumed at the village, historical records suggest a significant number of fins from harvested Tope Sharks were also likely being exported to China and other diaspora communities.
... All pre-PCR procedures (Decontamination, DNA extraction, and PCR setup) were conducted in a dedicated aDNA laboratory within the Department of Archaeology, Simon Fraser University (Burnaby, British Columbia, Canada), that is positively pressured and equipped with a UV HEPA air filter. Strict contamination control protocols, including the use of protective clothing, gloves, and regular cleaning of work surfaces with bleach, were followed throughout the analysis (Yang & Watt, 2005). No analyses of modern specimens or PCR amplifications have ever been performed within this laboratory. ...
... The quantity and quality of endogenous DNA preserved within ancient and historical remains is typically low, making them susceptible to contamination with modern DNA and amplification products (Cooper & Poinar, 2000;Yang & Watt, 2005). However, when taken together, various lines of evidence suggest we obtained authentic aDNA sequences from the archaeological short-tailed albatross specimens from Yuquot that we examined. ...
... Third, some of the sequences obtained from a handful of the the specimens exhibited C → T and G → A transitions associated with cytosine deamination, a form of post-mortem damage characteristic of aDNA (Dabney et al., 2013). Fourth, prior to DNA extraction, all of the remains were decontaminated through a combination of bleach and UV irradiation (Yang & Watt, 2005). Fifth, all pre-PCR procedures were conducted in a dedicated aDNA laboratory physically separated from the post-PCR laboratory (Cooper & Poinar, 2000). ...
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The short-tailed albatross (Phoebastria albatrus) is a threatened seabird whose present-day range encompasses much of the North Pacific. Within this species, there are two genetic clades (Clades 1 and 2) that have distinctive morphologies and foraging ecologies. Due to a global population collapse in the late 19th and early 20th centuries, the frequency of these clades among the short-tailed albatross population that historically foraged off British Columbia, Canada, is unclear. To document the species' historical genetic structure in British Columbia, we applied ancient DNA (aDNA) analysis to 51 archaeological short-tailed albatross specimens from the Yuquot site (Borden site number: DjSp-1) that span the past four millennia. We obtained a 141 bp cytochrome b sequence from 43 of the 51 (84.3%) analyzed specimens. Analyses of these sequences indicate 40 of the specimens belong to Clade 1, while 2 belong to Clade 2. We also identified a single specimen with a novel cytochrome b haplotype. Our results indicate that during the past four millennia most of the short-tailed albatrosses foraging near Yuquot belonged to Clade 1, while individuals from other lineages made more limited use of the area. Comparisons with the results of previous aDNA analyses of archaeological albatrosses from Japanese sites suggest the distribution of Clades 1 and 2 differed. While both albatross clades foraged extensively in the Northwest Pacific, Clade 1 albatrosses appear to have foraged along the west coast of Vancouver Island to a greater extent. Due to their differing distributions, these clades may be exposed to different threats.
... A commercial DNA extraction kit; DNA Investigating Kit (QIAGEN, Germany) also yielded DNA from old bones in this study (Fig 1.5a, and 1.5b). Contamination with other DNA sources is of the utmost concern when working [25,23] with old DNA . Contaminant human DNA can be introduced at any point during the processes of sampling, extraction and finally PCR setup [25,23,28,29] . ...
... Contamination with other DNA sources is of the utmost concern when working [25,23] with old DNA . Contaminant human DNA can be introduced at any point during the processes of sampling, extraction and finally PCR setup [25,23,28,29] . Therefore, strict laboratory procedures have been adhered to minimize such contaminations and this would affect the authenticity of the result of the DNA extract. ...
... Chandimal, K.M., Yasawardene, S.G., Ruwan, J. Illeperuma -Optimization of DNA extraction protocol using skeletal remains found in Sri Lanka Sri Lanka Anatomy Journal (SLAJ), 3(II) 2019,[16][17][18][19][20][21][22][23][24][25][26][27][28] ...
... Decontamination, DNA extraction, and PCR setup were all conducted in a dedicated aDNA laboratory in the Department of Archaeology, Simon Fraser University (Burnaby, BC, Canada) and followed strict contamination controls (Yang and Watt, 2005). In instances where samples were sufficiently large, only a portion of the individual bone was used for DNA extraction. ...
... Although archaeological fish remains often exhibit exceptional DNA preservation (Oosting et al., 2019), they, like all ancient skeletal remains, are highly susceptible to contamination from exogenous sources of modern DNA (Yang and Watt, 2005). However, various lines of evidence suggest our aDNA data are authentic rather than the result of systematic contamination. ...
... First, all pre-PCR laboratory work was conducted in a dedicated aDNA laboratory that is physically separated from modern DNA and post-PCR laboratories (Cooper and Poinar, 2000). Second, prior to DNA extraction, the samples were decontaminated using both bleach and UV irradiation (Yang and Watt, 2005). Third, no DNA was amplified from any of the blank extraction or negative PCR controls, indicating a lack of systematic contamination (Cooper and Poinar, 2000). ...
Article
Prior to European settlement, Indigenous peoples sustainably harvested Atlantic salmon (Salmo salar) and lake trout (Salvelinus namaycush) from Lake Ontario for centuries. Previous studies have suggested Indigenous peoples were able to maintain the productivity of Atlantic salmon and lake trout fisheries in the Great Lakes region through the use of resource management strategies. Since males tend to be the surplus sex among salmonids, one way in which Indigenous peoples could have managed Atlantic salmon and lake trout stocks was through the preferential harvesting of males. Here, we sought to investigate whether Indigenous peoples traditionally used sex-selective fishing to manage Lake Ontario Atlantic salmon and lake trout stocks. To address this question, we modified a DNA-based sex identification method developed for ancient Pacific salmonid (Oncorhynchus spp.) remains to make it applicable to archaeological Atlantic salmonid (Salmo spp.) and char (Salvelinus spp.) remains. This method assigns sex identities to samples through two PCR assays that co-amplify a fragment of the Y-specific salmonid master sex-determining gene (sexually dimorphic on the Y-chromosome gene) and an internal positive control, consisting of a fragment of the mitochondrial D-loop or nuclear clock1b gene. We applied this method to 61 Atlantic salmon and lake trout remains from the Antrex site (AjGv-38), a Middle Ontario Iroquoian (ca. CE 1250 to 1300) village located in the Lake Ontario watershed. Using this method, we successfully assigned sex identities to 51 of these remains (83.61% success rate), highlighting our method's sensitivity and efficacy. Statistical analyses indicate neither the aggregate sex ratio nor the sex ratios obtained for the individual species were male-biased. This suggests Antrex's Middle Ontario Iroquoian inhabitants probably did not practice male-selective fishing for Atlantic salmon or lake trout.
... A commercial DNA extraction kit; DNA Investigating Kit (QIAGEN, Germany) also yielded DNA from old bones in this study (Fig 1.5a, and 1.5b). Contamination with other DNA sources is of the utmost concern when working [25,23] with old DNA . Contaminant human DNA can be introduced at any point during the processes of sampling, extraction and finally PCR setup [25,23,28,29] . ...
... Contamination with other DNA sources is of the utmost concern when working [25,23] with old DNA . Contaminant human DNA can be introduced at any point during the processes of sampling, extraction and finally PCR setup [25,23,28,29] . Therefore, strict laboratory procedures have been adhered to minimize such contaminations and this would affect the authenticity of the result of the DNA extract. ...
... Chandimal, K.M., Yasawardene, S.G., Ruwan, J. Illeperuma -Optimization of DNA extraction protocol using skeletal remains found in Sri Lanka Sri Lanka Anatomy Journal (SLAJ), 3(II) 2019,[16][17][18][19][20][21][22][23][24][25][26][27][28] ...
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IntroductionThe preservation of DNA in old skeletal remains is reported to be very low in a tropical country like Sri Lanka due to prevailing climatic and environmental conditions such as high temperature, high rainfall and high humidity, etc. In this study, extraction of DNA from old skeletal remains dated back to 15 – 40 years was attempted by using previously published extraction protocols. Materials and MethodsA 15-years-old humerus (15Y) excavated from Kuliyapitiya area in Kurunegala district and the 40-yearsold tibia (40Y) received from Department of Anatomy, Faculty of Medical Sciences, University of Sri Jayewardenepura were used to extract old DNA. Human mitochondrial HVS I region of extracted DNA was amplified in PCR using four overlapping first round primers and second round nested primers respectively. A second-round nested PCR was performed. PCR amplification success was verified upon electrophoresis in 2% agarose gels. Results and AnalysisDNA bands were obtained with correct size ranges for all systems in both first and second round PCR products of amplified DNA extract of old bones 15Y and 40Y from modified phenolchloroform method. DNA bands were obtained from all four systems for 40Y bone DNA extract from DNA investigation Kit; QIAGEN, Germany. Conclusion In the present study, we have successfully extracted and amplified DNA from old skeletal remains by using modified phenol chloroform method and DNA investigation Kit – QIAGEN, Germany, nevertheless the preservation of DNA in skeletal remains in Sri Lanka is very low.
... Ancak insan aD-NA'sını konu alan çalışmalarda, hem kullanılan örneğin insana ait olması hem de çalışanın insan olması nedeni ile kontaminasyon riski diğerlerinden çok daha fazladır. 14,15 aDNA çalışmalarında kontaminasyonun önlenmesi ve ortadan kaldırılması ile ilgili en etkili yöntemler, önem sıralamaları değişmekle birlikte bu alanda yapılan farklı birçok çalışmada maddeler halinde sıralanmaktadır. Bu çalışmalara göre kontaminasyonun engellenmesi için alınan önlemleri iki başlık altında toplamak mümkündür. ...
... Bunlardan birincisi antik kalıntıların toplandığı kazı safhasında alınması gereken kontaminasyon önlemleri ve ikincisi aDNA'nın izole edilerek çoğaltıldığı laboratuvar içi kontaminasyon önlemleridir. 11,[13][14][15][16][17][18][19][20] I. Kazı Alanında Antik Malzemenin Toplanması Sürecinde Alınacak Kontaminasyon Önlemleri; 5 5. . Antik örnekler, içinde gerekli malzemelerin bulunduğu ''aDNA Numune Toplama Kiti'' yardımı ile toplanmalıdır. ...
... Kemik dokular kullanılırken bunlardan lezyonsuz olanlar seçilmelidir; çünkü lezyonlar kontaminasyon için ciddi bir kaynak oluştururlar. 15,17,21,[23][24][25] aDNA izole etmek için kaynak olarak yumuşak dokuların kullanılması durumunda, öncelikle yüzeyle teması olmayan ve kurumuş haldeki yumuşak doku örnekleri seçilmelidir. Kurumuş haldeki yumuşak dokuda sıvı oranının düşük olması dokuyu ve dolayısıyla da DNA'yı suyun ya da nemin neden olduğu hidrolitik zararlardan koruyabilir. ...
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The first biomarker used at the molecular level to distinguish humans from each other is the A, B, AB and O blood groups discovered by Karl Landsteiner in the early 20th century. The discoveries of the double-stranded structure of deoxyribonucleic acid (DNA) molecule, which contains all the necessary genetic code for survival of living things, by James Watson and Francis Crick in mid-century, and the polymerase chain reaction (PCR), which allowed the desired part of DNA to be amplified, by Kary B. Mullis in the last quarter of the same century, facilitated the development of new technologies and methods by creating very important leaps in molecular biology and genetics. These newly developed technologies and methods have made, by creating a locomotive force, very important contributions to molecular anthropology, forensic anthropology and forensic sciences, that have been developing in parallel with the fields of molecular biology and genetics. Ancient DNA sheds light on the molecular histories of living things, by using themethods and techniques developed in these fields and also by conceiving new techniques and methods specific to its specific field of study. Although there are some inherent difficulties in aDNA studies, contamination being the most emphasized, these studies make it easier to understand the evolution changes in many species, especially modern humans (Homo sapiens), over time by enlightening their molecular history. Evolution and phylogenetic relationships, identification, kinship analysis, gender determinations, the origins and spread of diseases, biological characteristics of extinct life forms and determination of migratory pathways of some species are the main topics of aDNA studies.
... Acidic conditions also increase the rate of DNA degradation, leading to poor DNA preservation in faunal remains from sites with acidic soils, such as those typical of the Boreal Forest (Allentoft et al. 2012;Burger et al. 1999;Lindahl and Nyberg 1972). As a result of DNA degradation, archaeological faunal remains are susceptible to contamination with modern DNA (Cooper and Poinar 2000;Yang and Watt 2005). Consequently, special precautions needed to be taken when conducting aDNA analysis. ...
... Consequently, special precautions needed to be taken when conducting aDNA analysis. These include extracting DNA in a genetic laboratory dedicated to analysing ancient materials, the use of protective clothing by lab workers, sample decontamination, the inclusion of controls to detect contamination, and the replication of results (Cooper and Poinar 2000;Yang and Watt 2005). ...
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More than a century of archaeological work in Ontario and surrounding areas has resulted in a massive quantity of archaeological data and collections, much of which has been subject to relatively limited analyses. For the past several years, our research team has been working on (re-)analyzing legacy faunal collections to understand past subsistence and economic activities, technologies, environments, and social relations at individual sites, while also contributing data and insights to larger, multi-site research projects that also use non-legacy data generated by ourselves and others. Drawing on this growing database of new and legacy zooarchaeological data, we have started to conduct largescale meta-analyses to explore broad trends across various aspects of the archaeology and historical ecology of the Lower Great Lakes region through roughly the past 1000 years. A theme that runs through all of this research is our desire to gain a better understanding of species that are now extinct, extirpated, or endangered. Through a combination of zooarchaeological meta-analysis and the use of modern analytical tools and techniques—including geographic information systems (GIS), stable isotope analysis, and ancient DNA (aDNA) analysis—our research is providing fascinating insights into past human interactions with, and the historical ecology of, such animals as the now-extinct passenger pigeon and the now-extirpated (locally extinct) Atlantic salmon. In this paper, as part of our commitment to sharing the results of our research with a wider audience, we provide a brief overview of some of our completed and ongoing research projects and publications that show how we use legacy faunal collections and legacy faunal data as a basis to explore various aspects of past human–animal relationships and the ecological history of these iconic species—and other species as well—in southern Ontario and the broader Great Lakes region.
... Contaminant DNA is a primary concern when handling aDNA, and the best way to minimize its impact is to take various precautionary measures in each step of sample processing [21,52]. Samples in this case study were processed by taking all the precautions listed below to minimize the contamination of endogenous aDNA. ...
... In addition, researchers should collect control samples to monitor contaminant DNA where samples were collected and stored (e.g., air, gloves, working benches, empty sampling tubes). These controls reflect the sampling environment and should be processed similarly to biological samples and considered during the bioinformatic analysis of samples [21,52]. ...
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High Throughput DNA Sequencing (HTS) revolutionized the field of paleomicrobiology, leading to an explosive growth of microbial ancient DNA (aDNA) studies, especially from environmental samples. However, aDNA studies that examine environmental microbes routinely fail to authenticate aDNA, examine laboratory and environmental contamination, and control for biases introduced during sample processing. Here, we surveyed the available literature for environmental aDNA projects—from sample collection to data analysis—and assessed previous methodologies and approaches used in the published microbial aDNA studies. We then integrated these concepts into a case study, using shotgun metagenomics to examine methodological, technical, and analytical biases during an environmental aDNA study of soil microbes. Specifically, we compared the impact of five DNA extraction methods and eight bioinformatic pipelines on the recovery of microbial aDNA information in soil cores from extreme environments. Our results show that silica-based methods optimized for aDNA research recovered significantly more damaged and shorter reads (<100 bp) than a commercial kit or a phenol–chloroform method. Additionally, we described a stringent pipeline for data preprocessing, efficiently decreasing the representation of low-complexity and duplicated reads in our datasets and downstream analyses, reducing analytical biases in taxonomic classification.
... All DNA extractions and PCR setups were conducted in a dedicated aDNA laboratory in the Department of Archaeology, Simon Fraser University (Burnaby, British Columbia). Vigorous contamination control protocols, including the regular cleaning of lab benches, separation of pre-and post-PCR laboratories, and the use of protective clothing, were adhered to throughout the analysis (Yang and Watt 2005). Prior to aDNA extraction, the samples were decontaminated using the protocol described by Speller et al. (2012). ...
... As a consequence of aDNA's degraded nature, ancient remains are susceptible to contamination with modern genomic DNA and PCR products (Yang and Watt 2005). Multiple lines of evidence indicate the DNA sequences obtained from the salmon vertebrae that yielded DNA are authentic and not the result of contamination. ...
Article
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This study uses ancient DNA analysis to identify the species of salmonids from a number of pre-contact Coast Salish settlements in Burrard Inlet, Canada dating from about 390 BCE to CE 1600. Our results indicate that chum salmon (Oncorhynchus keta) dominates all Burrard Inlet zooarchaeological assemblages through time, followed distantly by pink salmon (Oncorhynchus gorbuscha), sockeye (Oncorhynchus nerka), coho (Oncorhynchus kisutch), and Chinook (Oncorhynchus tshawytscha), indicative of very stable local fisheries. These results indicate that the four well-sampled sites appear to have been occupied during the fall and winter and perhaps during the spring.
... DNA retrieved from old and ancient substrates can be highly fragmented [37,[63][64][65][66][67][68][69][70][71][72]. Several factors contribute to the "natural" or induced breakdown of the double helix. ...
Article
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Diptera identification is fundamental in forensic entomology as well as in funerary archeoentomology, where the challenge is exacerbated by the presence of immature stages such as larvae and puparia. In these two developmental stages, specimens possess a very limited number of diagnostic features, and for puparia, there is also a lack of identification tools such as descriptions and identification keys. Morphological analysis, DNA-based techniques, and cuticular chemical analyses all show good potential for species identification; however, they also have some limitations. DNA-based identification is primarily hindered by the incompleteness of genetic databases and the presence of PCR inhibitors often co-extracted from the puparial cuticle. Chemical analysis of the cuticle is showing promising results, but this approach is also limited by the insufficient profile database and requires specific, expensive equipment, as well as trained personnel. Additionally, to ensure the repeatability of the analysis—a critical aspect in forensic investigations—and to preserve precious and unique specimens from museum collections, non-invasive protocols and techniques must be prioritized for species identification.
... Another important problem to consider during the microbial analysis of ancient samples is exogenous contamination. Contaminants can come from any step of the analysis, from the burial site to the transport and laboratory handling [204]. To avoid contamination, paleo-microbiological studies must be performed in dedicated clean laboratories, ideally exclusively dedicated to work on ancient remains and by following specific protocols to ensure the authenticity of the results [185,205]. ...
Article
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Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.
... We used ancient mitochondrial DNA (mtDNA) analysis to identify specimens from Palmrose (NISP ¼ 29), Tahkenitch Landing (NISP ¼ 1), and Par-Tee (NISP ¼ 14) to confirm ZooMS identifications or provide species-level identifications for those taxa where ZooMS identifications were restricted to the family level due to recent evolutionary divergence (Buckley et al. 2014). Ancient DNA analysis was conducted in two facilities: the Ancient DNA laboratory in the Department of Archaeology, Simon Fraser University, Burnaby and the AdaPT Facility at UBC following vigorous contamination control protocols (Yang and Watt 2005), including additional decontamination protocols (Speller et al. 2012). DNA was extracted using a modified silica-spin column method (Yang et al. 1998(Yang et al. , 2008. ...
Article
This study characterizes how Native Americans living on the Oregon coast used whales and small cetaceans prior to European contact. We present an original analysis of a large subsample (NISP ¼ 1177) of archaeological cetacean remains from the Palmrose (35CLT47) site, and new identifications from the previously analyzed Par-Tee (35CLT20; NISP ¼ 31) and Tahkenitch Landing (35DO130; NISP ¼ 33) sites. Using zooarchaeological and biomolecular analyses we report species presence and modification patterns to characterize use. Gray (Eschrichtius robustus) and humpback whale (Megaptera novaean-gliae) were the most commonly identified whale species and likely preferred source of food, oil, bone for tool manufacture, and possibly sinew. Dolphins and porpoises, especially harbor porpoise (Phocoena phocoena), were a source of food and possibly bone for tool manufacture. While site inhabitants may have engaged in opportunistic hunting, the presence of species such as blue (Balaenoptera muscu-lus) and Cuvier's beaked whale (Ziphius cavirostris) suggests collection of beached animals was also an important acquisition strategy. Our study demonstrates the value of: (1) biomolecular analyses for improved species identifications and understanding of species richness ; and (2) thorough zooarchaeological analyses to fully understand dietary and cultural contributions of cetaceans to pre-contact lifeways on the Oregon coast. ARTICLE HISTORY
... All DNA samples were extracted and processed in a dedicated ancient DNA facility at the University of Kiel following the guidelines on contamination control in ancient DNA [57][58][59], according to a previously published protocol for the non-UGD treated samples [60]. Shotgun sequencing was performed on the Illumina HiSeq 6000 (2 × 100) platform of the Institute of Clinical Molecular Biology in Kiel. ...
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Yersinia pestis is the causative agent of at least three major plague pandemics (Justinianic, Medieval and Modern). Previous studies on ancient Y. pestis genomes revealed that several genomic alterations had occurred approximately 5000–3000 years ago and contributed to the remarkable virulence of this pathogen. How a subset of strains evolved to cause the Modern pandemic is less well-understood. Here, we examined the virulence-associated prophage (YpfΦ), which had been postulated to be exclusively present in the genomes of strains associated with the Modern pandemic. The analysis of two new Y. pestis genomes from medieval/early modern Denmark confirmed that the phage is absent from the genome of strains dating to this time period. An extended comparative genome analysis of over 300 strains spanning more than 5000 years showed that the prophage is found in the genomes of modern strains only and suggests an integration into the genome during recent Y. pestis evolution. The phage-encoded Zot protein showed structural homology to a virulence factor of Vibrio cholerae. Similar to modern Y. pestis, we observed phages with a common origin to YpfΦ in individual strains of other bacterial species. Our findings present an updated view on the prevalence of YpfΦ, which might contribute to our understanding of the host spectrum, geographical spread and virulence of Y. pestis responsible for the Modern pandemic.
... As Raff [27] reports, aDNA research cannot be done in regular molecular biology laboratories. While next-generation methods have made it much easier to distinguish contamination from endogenous DNA based on DNA damage patterns, preventing contamination during the extraction process is still extremely difficult (e.g., [31,32]). Contamination is such a pervasive problem that the work requires specialised facilities and workers trained in aDNA protocols. ...
Article
Proteomics has become an attractive method to study human and animal material, biological profile, and origin as an alternative to DNA analysis. It is limited by DNA amplification in ancient samples and its contamination, high cost, and limited preservation of nuclear DNA. Currently, three approaches are available to estimate sex–osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. Proteomics provides a new, seemingly simple, and relatively non-expensive way of sex estimation without the risk of contamination. Proteins can be preserved in hard teeth tissue (enamel) for tens of thousands of years. It uses two sexually distinct forms of the protein amelogenin in tooth enamel detectable by liquid chromatography-mass spectrometry; the protein amelogenin Y isoform is present in enamel dental tissue only in males, while amelogenin isoform X can be found in both sexes. From the point of view of archaeological, anthropological, and forensic research and applications, the reduced destruction of the methods used is essential, as well as the minimum requirements for sample size.
... DNA analysis from archaeological human samples was conducted in a clean environment in the aDNA facility at the Interdisciplinary Research Institute on Bio-Nano-Sciences, Babeș-Bolyai University, Romania. All experimental procedures, particularly DNA extraction and PCR amplifications, were performed following strict protocols to ensure the authenticity of the data (Roberts and Ingham, 2008, Pääbo et al., 2004, Yang and Watt, 2005. ...
Article
Objective: This study attempts to integrate multiple methods to investigate the presence of malaria in human skeletal samples from an archaeological context. Materials: 33 well preserved human remains originating from a 17th-century archaeological site in southeastern Romania. Methods: The human bone samples were analyzed using rapid diagnostic tests for malaria antigens and PCR amplification of Plasmodium falciparum apical membrane antigen 1. A preliminary test was performed to identify and briefly characterize the presence of hemozoin using a combination of TEM imaging and diffraction. Results: The rapid diagnostic tests indicated that more than half of the examined samples were positive for Plasmodium antigens, but no traces of the parasites' genetic material were detected despite repeated attempts. The TEM images indicated that hemozoin might be a promising diagnostic marker of malaria in ancient bones. Conclusions: The indisputable identification of malaria in the analyzed archaeological population was not possible as none of the applied methodological strategies turned out to be straightforward. Significance: This study reinforces the intricacy and limitations of unequivocally identifying malaria in past populations and sets the stage for future studies on such life-threatening infectious disease in a geographical space, which is currently underrepresented in the bioarchaeological literature. Limitations: The low sample size and the lack of consistency across all assays hindered understanding the role of malaria in the studied population. Suggestions for further research: Further thorough multidisciplinary approaches on malaria detection in ancient settlements would be appropriate to inform our knowledge of its origins, frequency, and pathogen changes over centuries.
... DNA extraction of archaeological samples. All sample preparations and aDNA extractions were conducted in a dedicated aDNA laboratory at Simon Fraser University, Canada, following strict protocols to minimize contamination with modern DNA 54 . Bones were chemically decontaminated through immersion in 6% sodium hypochlorite solution for 5 min. ...
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Phenological diversity in food resources prolongs foraging opportunities for consumers and buffers them against environmental disturbances. Such diversity is particularly important in forage fish such as Pacific herring (Clupea pallasii), which are foundational to coastal food webs and fisheries. While the importance of phenological diversity is well-known from contemporary studies, the extent to which different populations contribute to fisheries over long time scales is mostly unknown. In this study, we investigated the relative contributions of genetically and phenologically distinct herring populations to Indigenous Peoples’ food systems over multiple centuries, using ancient DNA extracted from archaeological herring bones. These bones were excavated from two Coast Salish archaeological sites (Burton Acres Shell Midden and Bay Street Shell Midden) in the Puget Sound region, USA. Using genetic stock identification from seven nuclear DNA markers, we showed that catches at the two sites in central Puget Sound were dominated by January–February and March–April spawners, which are the contemporary spawning groups in the vicinity of the sites. However, May spawners were detected in the older Burton Acres assemblage (dated to 910–685 cal BP), and a mixed stock analysis indicated that catches at this site consisted of multiple populations. These results suggest that Coast Salish ancestors used a portfolio of herring populations and benefited from the ecological resource wave created by different spawning groups of herring. This study of ancient DNA allowed us to glimpse into Indigenous traditional food and management systems, and it enabled us to investigate long-term patterns of biodiversity in an ecologically important forage fish species.
... Species-level identifications were previously assigned to these samples through aDNA analysis by Morin et al. 38 conducted in a dedicated ancient DNA laboratory in the Department of Archaeology, Simon Fraser University (Burnaby, BC, CA), that is physically separated from the post-PCR laboratory. Rigorous contamination control protocols were followed throughout the analysis 58 . All the samples were decontaminated prior to DNA extraction through a combination of bleach washes and UV irradiation 59 . ...
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To gain insight into pre-contact Coast Salish fishing practices, we used new palaeogenetic analytical techniques to assign sex identifications to salmonid bones from four archaeological sites in Burrard Inlet ( Tsleil-Waut ), British Columbia, Canada, dating between about 2300–1000 BP (ca. 400 BCE–CE 1200). Our results indicate that male chum salmon ( Oncorhynchus keta ) were preferentially targeted at two of the four sampled archaeological sites. Because a single male salmon can mate with several females, selectively harvesting male salmon can increase a fishery’s maximum sustainable harvest. We suggest such selective harvesting of visually distinctive male spawning chum salmon was a common practice, most effectively undertaken at wooden weirs spanning small salmon rivers and streams. We argue that this selective harvesting of males is indicative of an ancient and probably geographically widespread practice for ensuring sustainable salmon populations. The archaeological data presented here confirms earlier ethnographic accounts describing the selective harvest of male salmon.
... A study on the extent of modern contamination was presented by Malmstr€ om, investigating animal remains for human DNA: all 29 samples contained human DNA, but in 25 cases authentic animal DNA was found also (Malmstr€ om et al., 2005). To eliminate contamination, procedures were developed to assure the collection of authentic ancient DNA (Yang and Watt, 2005;Bouwman et al., 2006;Anderung et al., 2008). To ensure authentic and credible data they suggest: ...
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For centuries, ancient Egyptian Royal mummies have drawn the attention both of the general public and scientists. Many royal mummies from the New Kingdom have survived. The discoveries of the bodies of these ancient rulers have always sparked much attention, yet not all identifications are clear even nowadays. This study presents a meta-analysis to demonstrate the difficulties in identifying ancient Egyptian royal mummies. Various methods and pitfalls in the identification of the Pharaohs are reassessed since new scientific methods can be used, such as ancient DNA-profiling and CT-scanning. While the ancestors of Tutankhamun have been identified, some identities are still highly controversial (e.g., the mystery of the KV-55 skeleton, recently most likely identified as the genetic father of Tutankhamun). The meta-analysis confirms the suggested identity of some mummies (e.g., Amenhotep III, Thutmo-sis IV, and Queen Tjye). Am J Phys Anthropol 159:S216-S231,
... Throughout the molecular procedures, special precautions were taken to avoid contamination including using sterile tubes, filtered pipette tips, freshly prepared reagents, and pipettes not previously exposed to plant DNA. Samples from historical nests were physically isolated from contemporary nest samples in storage and lab procedures to prevent cross-sample contamination [28]. PCR reactions were performed in 50 μL volume containing 2 μL of DNA template, 25mM MgCl2, 1x Standard Taq Buffer, 2.5mM of each dNTP, 1uM of the forward and reverse primers [24], and 1 unit of Taq DNA polymerase (New England Biolabs, Ipswich, MA). ...
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Bird nests in natural history collections are an abundant yet vastly underutilized source of genetic information. We sequenced the nuclear ribosomal internal transcribed spacer to identify plant species used as nest material in two contemporary (2003 and 2018) and two historical (both 1915) nest specimens constructed by Song Sparrows (Melospiza melodia) and Savannah Sparrows (Passerculus sandwichensis). A total of 13 (22%) samples yielded single, strong bands that could be identified using GenBank resources: six plants (Angiospermae), six green algae (Chlorophyta), and one ciliate (Ciliophora). Two native plant species identified in the nests included Festuca microstachys, which was introduced to the nest collection site by restoration practitioners, and Rosa californica, identified in a nest collected from a lost habitat that existed about 100 years ago. Successful sequencing was correlated with higher sample mass and DNA quality, suggesting future studies should select larger pieces of contiguous material from nests and materials that appear to have been fresh when incorporated into the nest. This molecular approach was used to distinguish plant species that were not visually identifiable, and did not require disassembling the nest specimens as is a traditional practice with nest material studies. The many thousands of nest specimens in natural history collections hold great promise as sources of genetic information to address myriad ecological questions.
... Moisture leads to hydrolytic and oxidative damage in DNA. Bones are porous and when exposed to moisture, H2O accumulates in the pores and creates favorable conditions for the growth of microorganisms leading to contamination as well as degradation (36,37,38). In addition to humidity and temperature, soil type and pH are also parameters need to be considered in aDNA studies. ...
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Life gave rise on our planet 3-4 billion years ago and since then, living organisms (from one cell to multicellular organisms) have undergone many genetic, phenotypic and communal changes. Scientists have been able to shed light on only a small part of this evolutionary process, but with the development of new techniques our knowledge is expanding day by day. For the past 30 years ancient DNA studies have aided us in understanding the molecular basis of the changes observed in living organisms. Ancient DNA (aDNA) is the genetic material obtained from biological remains (bones, teeth, plant seeds, etc.) acquired from archaeological and paleontological excavations. In the present review, molecular studies carried out to date, contributions of ancient DNA to medical sciences, as well as basic problems encountered in obtaining and using aDNA have been discussed.
... Postexcavation sample treatment and storage conditions can also affect DNA preservation and introduce contamination. For example, washing specimens after excavation can both introduce contaminating DNA and simultaneously induce hydrolytic damage to endogenous DNA (Llamas et al. 2017b;Yang and Watt 2005). Thus, at the best of times, a researcher may expect an ancient sample to be mostly composed of environmental and microbial contamination (99 percent) with low quantities of poorly preserved endogenous DNA (<1 percent) (Carpenter et al. 2013). ...
... Paleomicrobiological studies on ancient molecules must follow standard protocols to avoid modern exogenous contamination and ensure the authenticity of the results [30]. Contaminating molecules can come from the burial site, handling, transport, storage, and laboratory environments; from the materials and instruments used during the analysis; and from the operators at each step of the study [31,32]. Regardless of the starting sample type, all the steps of the analysis of ancient molecules must be performed in a dedicated clean hood and laboratory, ideally exclusively dedicated to work on ancient remains, excluding any positive controls, and including several negative controls that should be handled strictly in parallel with investigated samples [31]. ...
Article
The microbiota is a hot topic of research in medical microbiology, boosted by culturomics and metagenomics, with unanticipated knowledge outputs in physiology and pathology. Knowledge of the microbiota in ancient populations may therefore be of prime interest in understanding factors shaping the coevolution of the microbiota and populations. Studies on ancient human microbiomes can help us understand how the community of microorganisms presents in the oral cavity and the gut was shaped during the evolution of our species and what environmental, social or cultural changes may have changed it. This review cumulates and summarizes the discoveries in the field of the ancient human microbiota, focusing on the remains used as samples and techniques used to handle and analyze them.
... Protocols for minimizing contamination during handling of the skeletal remains recovered from the burial in Bowie County, Texas were in alignment with contamination prevention measures standardly recommended for archaeological and ancient DNA specimens, including: (a) use of protective suits, gloves, masks, hairnets; (b) bleach decontamination and UV-irradiation of work benches and associated equipment; (c) physical removal and/or chemical destruction of contaminant DNA on external bone surfaces; (d) extraction of bone samples in a designated low-copy area; (e) PCR amplification in a location that is physically separated from the extraction area; (f) use of appropriate negative controls, reagent blanks, positive controls; and (g) replicate testing [16][17][18][19][20][21]. ...
Article
In 1932, seven burials were discovered on a Texas plantation that was originally the site of a 17 th -century Caddo Indian village. Of the seven excavated graves, one set of remains (an adult male) was notably buried in a manner inconsistent with traditional Caddoan burial practices and has long been purported to be the remains of Sieur de Marle (a member of the French explorer La Salle’s last expedition). Diary accounts of La Salle’s expedition scribe report that Sieur de Marle died along a river near an Indian village during a trek to Canada to find help for colonists left behind at the ill-fated Fort St. Louis. Additionally, two lead projectiles recovered from the grave were ballistically analyzed and determined to be consistent with ammunition used in 17 th century weaponry. In the 1980s, anthropologists requested access to the remains for study, but the skull was missing. Cranial measurements recorded in 1940 and 1962 (by two independent anthropologists) were used to investigate the ancestry of this individual; and the Giles-Elliot (G-E) discriminant function was calculated to be 18.1, within the Anglo-European range. Dietary isotope testing on non-cranial skeletal elements determined that this unknown male’s diet was rich in animal/marine protein sources, which differs appreciably from Caddo Indian populations of that time period. In order to genetically assess this individual’s biogeographic ancestry and to provide further support that this individual is of European descent, mitochondrial DNA (mtDNA) sequencing was performed using the Applied Biosystems ™ Precision ID mtDNA Whole Genome Panel. mtDNA sequencing of multiple sections from two different long bones yielded compiled results consistent with Haplogroup H, a predominantly European mtDNA haplogroup. Further anthropological calculations were conducted using cranial measurements, FORDISC™ software, and discriminant function analysis. Two-way, four-way, and multigroup discriminant function analyses further classify this set of unidentified remains as being White (European) in origin, with posterior probabilities of 0.999, 0.881 and 0.986, respectively. Combined with historical records of Sieur de Marle’s death, as well as overlays of historical and contemporary maps which demonstrate that the plantation site aligns with Joutel’s diary accounts of de Marle’s burial, these collective results support that these remains are of a European male and may possibly belong to this prominent member of La Salle’s expedition team.
... 44,45 Finally, the survival of mitochondrial DnA in ancient bones has been known for several decades, but authentication and contamination have been major issues. 46,47 More recently, technological developments have led to reliable information being obtained by DnA analysis, in terms of gender identification, parental relationships and past people connections, 48,49 which have produced significant advances in our historical knowledge. ...
Chapter
Reviewing the analytical strategies used in the study of cultural heritage assets such as movable artworks and archaeological items, and immovable objects like mural paintings, archaeological sites and historical buildings, this book pays particular attention to analytical methodology. It is not always necessary to use new and sophisticated instrumentation, what is important is how the instruments are used to obtain reliable, reproducible and repetitive results in view of the problems to be solved. The book considers the influence of the environment on the conservation state including degradation and how modern analytical methods have improved the analysis of materials. It emphasizes multi-method approaches on a range of materials, an approach that is of keen interest to those working in conservation practice. Primarily aimed at final year undergraduate study and masters level students, it would also be useful as supplementary reading for postgraduates and academics who require analytical techniques to enhance their research.
... Archaeological tooth dentine was sampled at the Laboratories of Molecular Anthropology and Microbiome Research (LMAMR) at the University of Oklahoma, Norman, in the dedicated sample preparation area following standard ancient DNA contamination protocols [59,60]. Dental calculus was sampled on location in museum research collections following a calculus-specific sampling protocol designed to reduce contamination (description in the electronic supplementary material with photos/specimen metadata). ...
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Genetic analyses are an important contribution to wildlife reintroductions, particularly in the modern context of extirpations and ecological destruction. To address the complex historical ecology of the sea otter ( Enhydra lutris ) and its failed 1970s reintroduction to coastal Oregon, we compared mitochondrial genomes of pre-extirpation Oregon sea otters to extant and historical populations across the range. We sequenced, to our knowledge, the first complete ancient mitogenomes from archaeological Oregon sea otter dentine and historical sea otter dental calculus. Archaeological Oregon sea otters ( n = 20) represent 10 haplotypes, which cluster with haplotypes from Alaska, Washington and British Columbia, and exhibit a clear division from California haplotypes. Our results suggest that extant northern populations are appropriate for future reintroduction efforts. This project demonstrates the feasibility of mitogenome capture and sequencing from non-human dental calculus and the diverse applications of ancient DNA analyses to pressing ecological and conservation topics and the management of at-risk/extirpated species.
... One possible reason for a lack of decontamination was physical contact of the clean interior pieces with contaminated materials and dust generation during the subsampling. We noted that our test plates were contaminated with tracers and other cells during disk sampling methods, likely indicating the production of contaminated dust or aerosols during processing, similar to previous findings 25,56 . Thus, methods that minimize dust and aerosol generation are recommended to decrease the possibility of re-contaminating cleaned samples. ...
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This study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. In addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost researchers to acquire contaminant-free DNA from permafrost with low biomass.
... [2,29,30] Several precautions can guard against contamination. [17,31,32] Protective clothing during excavation [33,34] and lab work minimizes the introduction of contaminating DNA, and the irradiation of reagents, lab equipment, and clean room facilities are often used to degrade DNA from other potential sources. [35,36] Despite these efforts, a low level of contamination is unavoidable, and contamination is therefore closely monitored by using negative controls during DNA extraction and library preparation [37] and by the inclusion of unique combinations of DNA barcodes in each ancient DNA library. ...
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Present‐day contamination can lead to false conclusions in ancient DNA studies. A number of methods are available to estimate contamination, which use a variety of signals and are appropriate for different types of data. Here an overview of currently available methods highlighting their strengths and weaknesses is provided, and a classification based on the signals used to estimate contamination is proposed. This overview aims at enabling researchers to choose the most appropriate methods for their dataset. Based on this classification, potential avenues for the further development of methods are discussed. To ensure that ancient DNA studies yield valid results, it is crucial to quantify present‐day DNA contamination. The signals and methods that can be used to estimate the proportion of contamination are surveyed.
... The authors are aware that the PCR-based approach is currently at stake for ancient DNA analysis, due to the outstanding developments of nextgeneration sequencing techniques (Llamas et al. 2017). In spite of this advice, this basic approach is simple and costeffective and the use of sterile environment, reagents, and contamination precautions (Yang and Watt 2005) could provide preliminary results to be confirmed by more timeconsuming and expensive NGS protocols (Fu et al. 2016). ...
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Recent strontium isotope (87Sr/86Sr) analyses of bones and teeth have provided useful archeological results for reconstructing past human migration and diet. We report 87Sr/86Sr ratios and DNA analyses of tooth enamel from individuals buried in some necropolises in Nola town, near Napoli (Campania, South Italy). These individuals lived in the period between the Avellino (1925 years BCE) and CE 472 Pollena Vesuvian eruptions and are dated on archeological basis to the time span between the sixth and second century BCE. Tooth enamel 87Sr/86Sr ratios (0.70788–0.70864) are higher than baseline values in the necropolises (0.70756–0.70792): this can be explained by assuming either that all the analyzed individuals are not local—an unlikely possibility—or that they ate both local and foreign food (within about 50 km), including 87Sr-rich seafood. An explanation for such a varied diet might be that the individuals from Nola were living near the Ancient Appia and Popilia ways and not far from the coastline. Whatever its origin, the 87Sr/86Sr ratios represent the isotopic signature of the local community living on the slopes of Mt. Somma-Vesuvius between the sixth and second centuries BCE. This knowledge will support future isotope studies on volcanic eruptions as possible causes of human migration.
... Sample decontamination, DNA extraction, and PCR setup were all performed in a dedicated ancient DNA laboratory in the Department of Archaeology at Simon Fraser University (Burnaby, BC, Canada). To reduce the likelihood of contamination, strict contamination control protocols, including the separation of the pre-PCR and post-PCR laboratories, the use of protective clothing, and the regular cleaning of work surfaces with bleach, were adhered to throughout the analysis (Cooper and Poinar, 2000;Yang and Watt, 2005). Ancient DNA analysis was conducted on 11-57 mg subsamples of bone removed from each vertebra. ...
Article
Prior to their extirpation around 1900 CE, Lake Ontario hosted the world’s largest freshwater Atlantic salmon (Salmo salar) fishery. Due to their early disappearance, questions remained about fundamental aspects of the species’ biology, such as whether they belonged to sea-run (anadromous) or freshwater resident (potamodromous) ecotypes. Recent isotopic analyses have demonstrated that the complex of Atlantic salmon populations spawning in tributaries emptying along Lake Ontario’s northern shores were potamodromous. However, no evidence has yet been gathered for Atlantic salmon migratory behaviour from Lake Ontario’s southeastern region, where historical observations suggest both anadromous and potamodromous populations may have spawned. Here, we provide the first results for isotopic analyses of bone collagen from seven fish bones from archaeological sites (c. 1427 to 1600 CE) identified as Atlantic salmon through ancient DNA and zooarchaeological analyses. The results of the isotopic analyses confirm that at least some of the salmon spawning in tributaries emptying into Lake Ontario’s southeastern shores were also potamodromous. Although further analyses are needed, this suggests anadromy may have been completely absent in Lake Ontario’s complex of Atlantic salmon populations in recent centuries.
... Sample processing, DNA extraction and DNA library preparation have previously been described in Krause-Kyora et al. 24 . DNA extractions and pre-PCR steps were performed in clean room facilities dedicated to aDNA research, following the guidelines on contamination control in aDNA studies [52][53][54] . For each sample, two different double-stranded DNA sequencing libraries (UDG-treated and non-UDG-treated) were prepared. ...
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The highly polymorphic human leukocyte antigen (HLA) plays a crucial role in adaptive immunity and is associated with various complex diseases. Accurate analysis of HLA genes using ancient DNA (aDNA) data is crucial for understanding their role in human adaptation to pathogens. Here, we describe the TARGT pipeline for targeted analysis of polymorphic loci from low-coverage shotgun sequence data. The pipeline was successfully applied to medieval aDNA samples and validated using both simulated aDNA and modern empirical sequence data from the 1000 Genomes Project. Thus the TARGT pipeline enables accurate analysis of HLA polymorphisms in historical (and modern) human populations.
... All pre-PCR procedures (decontamination, DNA extraction, and PCR setup) were conducted at the Department of Archaeology at Simon Fraser University (Burnaby, BC, Canada), in a dedicated aDNA laboratory and followed strict contamination control protocols [127]. All of the analyzed samples were decontaminated prior to DNA extraction using established protocols [128]. ...
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The ability to distinguish between different migratory behaviours (e.g., anadromy and potamodromy) in fish can provide important insights into the ecology, evolution, and conservation of many aquatic species. We present a simple stable carbon isotope (δ¹³C) approach for distinguishing between sockeye (anadromous ocean migrants) and kokanee (potamodromous freshwater residents), two migratory ecotypes of Oncorhynchus nerka (Salmonidae) that is applicable throughout most of their range across coastal regions of the North Pacific Ocean. Analyses of kokanee (n = 239) and sockeye (n = 417) from 87 sites spanning the North Pacific (Russia to California) show that anadromous and potamodromous ecotypes are broadly distinguishable on the basis of the δ¹³C values of their scale and bone collagen. We present three case studies demonstrating how this approach can address questions in archaeology, archival, and conservation research. Relative to conventional methods for determining migratory status, which typically apply chemical analyses to otoliths or involve genetic analyses of tissues, the δ¹³C approach outlined here has the benefit of being non-lethal (when applied to scales), cost-effective, widely available commercially, and should be much more broadly accessible for addressing archaeological questions since the recovery of otoliths at archaeological sites is rare.
... In instances where specimens were genetically identified as passenger pigeons, we sought to assign them to one of the two passenger pigeon haplogroups, termed Haplogroup 1 and 2 (Novak, 2016), identified by Murray et al. (2017). All decontamination, DNA extraction, and PCR setup procedures were conducted in a dedicated aDNA laboratory at the Department of Archaeology, Simon Fraser University (Burnaby, BC), and incorporated strict contamination controls (Yang and Watt, 2005). Following Speller et al. (2012), all specimens were decontaminated prior to DNA extraction through a combination of submersion in bleach and UV irradiation. ...
Article
The decline of passenger pigeons (Ectopistes migratorius) during the late nineteenth century continues to draw substantial public and scientific attention as perhaps the most (in-)famous extinction event in North America's recent history. While humans undeniably caused the extinction, the relative importance of indirect (habitat destruction) versus direct (overhunting) impacts has remained a mystery, in part, due to a lack of scientific evidence for critical aspects of the species' dietary ecology. One key factor in explaining why passenger pigeons went extinct is that their highly specialized diet and foraging strategy, focusing on mast (tree nuts), were no longer feasible as the forested habitats that they depended on became depleted by deforestation. We used stable isotope (d 13 C and d 15 N, n=94) and ancient DNA (n=9) analyses of archaeological specimens to demonstrate that, during the later Holocene, passenger pigeons had a substantial degree of dietary plasticity (including some individuals specializing in consumption of agricultural crops) that could have allowed them to take advantage of other food opportunities when mast became scarce. Dietary variation is not linked with either biological age (juveniles versus adults) or haplogroup. These results suggest that habitat destruction was less important for the passenger pigeon's extinction than the impacts of hunting and trapping and highlight the tremendous potential of the archaeological record for exploring the factors that led to this species' extinction.
... Bone collagen samples obtained from faunal remains, mainly bison, from Zone II and 128 Subzone IIIa have produced numerous radiocarbon dates (Table 1). Some specimens of bison 129 were dated more than once; in these cases we provide both dates in All pre-PCR procedures were conducted in a dedicated ancient DNA laboratory in the 157 Department of Archaeology, Simon Fraser University, and followed strict contamination control 158 protocols (Yang and Watt 2005). Prior to DNA extraction, all specimens were decontaminated 159 using the protocol described by Speller et al. (2012). ...
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Ancient DNA was extracted from 12 500 to 10 500 year old ground squirrel bones from Tse’K’wa, an archaeological site in the Peace River region of northeastern British Columbia, Canada. Analysis of mitochondrial DNA from seven individuals demonstrates that all are Urocitellus richardsonii (Richardson’s ground squirrel), a species not found in the region today. Phylogenetic and sequence analyses indicate these individuals share a previously undocumented mitochondrial control region haplotype that is most closely related to haplotypes observed in modern specimens from Saskatchewan and Montana. At the end of the Pleistocene these ground squirrels extended their range north and west into open vegetation communities that developed when ice sheets melted and glacial lakes drained. They were subsequently extirpated from the Peace River region when forests replaced earlier pioneering vegetation communities.
... Tools and surfaces that are likely to be in contact with samples should be cleaned with diluted bleach. It is also important that the samples are not washed, since even purified water often contains DNA from exogenous sources that can permeate the excavated sample (Yang and Watt 2005). ...
Chapter
Genetic sequences have traditionally been generated solely from modern individ- uals. Advances in laboratory and sequencing techniques, however, have made it possible to retrieve genetic information from fossil, archaeological, museum, or otherwise dead and degraded specimens. Genetic material derived from ancient specimens is referred to as ancient DNA (aDNA). The main advantage of ancient DNA is that it allows researchers to study past genetic diversity directly, rather than having to rely on modern genetic patterns to infer past population processes. Genetic sequences generated from ancient samples have been successfully employed to tackle long-standing questions in the fields of archaeology and genet- ics. Ancient DNA has been especially important for reconstructing population histories, for example, to study past population interactions and relationships between past and present populations and species; to quantify past levels of movement and identify major migration episodes; as well as to investigate where and when different plant and animal species were domesticated. Ancient DNA has also been used to calibrate molecular clocks that measure the rates of evolution in different species (e.g., Palkopoulou et al. 2015; Rieux et al. 2014; Skoglund et al. 2015) and to date past demographic events, such as population splits, expansions, and fluctuations in population size (particularly in the recent past), as well as to generate date estimates for previously undated specimens. Ancient DNA can also be used for species identification (e.g., Horsburgh 2008); for establishing the biological sex of ancient individuals (Skoglund et al. 2013); and for reconstructing past environments and past diets. The ability to extract data from archaeological specimens opens inroads to research areas previously inaccessible. For example, ancient DNA has allowed researchers to directly estimate past phenotypes and to study the effect of changing environments on genetic variation (e.g., Girdland Flink et al. 2014; Krause et al. 2007; Lalueza-Fox et al. 2007; Ludwig et al. 2009; Olalde et al. 2014) and, by measuring changes in the frequency of adaptive genetic variants over time, researchers have been able to directly estimate the strength of natural selection at different genetic loci (genes) (Loog et al. 2017; Ludwig et al. 2009; Sverrisdóttir et al. 2014; Wilde et al. 2014). The retrieval of ancient DNA does not come without challenges. After the death of an organism, the DNA in their cells starts rapidly degrading due to internal and external factors, inevitably resulting in a complete lack of recoverable DNA. In favorable conditions, however, DNA can survive up to hundreds of thousands of years (e.g., Orlando et al. 2013). Nevertheless, the recovered ancient DNA molecules tend to be fragmented, damaged, and at risk of contamination by modern-day sources. This reduction in quantity and quality requires specific techniques to retrieve and analyse ancient DNA.
... Second, the modern beluga samples as a contamination source was excluded by receiving and processing them in a separate laboratory after the analysis of the 19th century samples had been completed. Third, the samples were decontaminated through a combination of physical abrasion, UV irradiation, as well as bleach, HCl, and NaOH washes (Yang & Watt, 2005). Fourth, the failure to amplify DNA from any of the blank extraction and negative PCR controls indicates there was a lack of systematic contamination (Poinar, 2003). ...
Article
Marine mammals often exhibit significant sexual segregation in their diet and habitat use but these differences have not been studied systematically in historic or ancient populations due to the difficulties associated with determining the sex of skeletal elements based on gross morphology. Using a combined ancient DNA and stable isotope approach, we document a sexual difference in the foraging ecology of late 19th century beluga whales (Delphinapterus leucas) from the Canadian High Arctic. Using two PCR assays that coamplify fragments of the Y‐linked SRY and X‐linked ZFX genes, we assigned reproducible sex identities to 35 beluga specimens. This provided a basis for investigating sex‐specific differences in foraging ecology using stable carbon and nitrogen isotope analyses of bone collagen. These isotopic data demonstrate that although both males and females primarily consumed Arctic cod, males utilized a wider range of prey than females, feeding on high trophic level benthic prey (sculpins) to a greater extent. Because bone collagen integrates prey isotopic compositions over the course of several years these sex‐based differences in beluga bone collagen isotopic compositions reflect long‐term and sustained sexual differences in foraging.
... Postmortem degradation of DNA is a complex phenomenon, beginning with autolysis and followed by aerobic and bacterial destruction of the cells (Ludes et al., 1993). Physical and chemical degradation can destroy most of the DNA (Yang and Watt, 2005). Physical influences may include humidity, irradiations, photoelectric influences, but the most frequent physical influence has been always the temperature (Richardson et al., 2006). ...
Article
This study presents zooarchaeological and ancient DNA (aDNA) analyses of the remains of a non-local gopher tortoise (Gopherus polyphemus) recovered from a ca. 1850s–1861 privy in New Orleans, Louisiana. This tortoise pre-dates the known import of these animals to the city beginning in the 1870s, raising questions about how and why this individual was brought to New Orleans. To address these questions, we use a median-joining network and maximum-likelihood tree to identify the haplogroup to which the archaeological tortoise likely belonged, and we combine these results with historical data to suggest a likely origin point in the western portions of the species' range. We place these findings within the fabric of the historical development of New Orleans, which nearly tripled in population from 1830 to 1860 and which saw a concomitant import of a diverse array of foods to meet consumer demand. Ultimately, we argue that the gopher tortoise is likely the result of entrepreneurialism, experimentation, and creative attempts to develop reliable food supply that fit within New Orleans’ cuisine.
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Adli biyoloji, adli soruşturmalarda olay yerinden toplanan biyolojik kanıtların analizi ve değerlendirilmesiyle ilgilenen bir bilim dalı olup biyolojinin birçok alt dalını barıdıran geniş bir yelpazaye sahiptir. Her türlü biyolojik kanıtların toplanması, korunması, analizi ve yorumlanmasınında adalate katkıda bulunur. Bu kitap, adli bilimlerde lisans, yüksek lisans ve doktora eğitimi alan öğrencilerin adli biyoloji alanında yararlanabilecekleri iki ciltten oluşan kapsamlı bir kaynaktır. Kitapta, adli biyolojinin farklı alanlarına odaklanarak, uzman yazarlar tarafından kaleme alınmış bölümlerle zenginleştirilmiştir. Kitabın birinci cildinde ele alınan konu başlıkları adli bilimlerde hayvanlar ve entomolojik izler: Adli bilimlerde evcil ve yaban hayvanlar ve adli entomoloji, bitkilerin gölgesindeki sırlar: adli botanik, adli bilimlerde zehir ve/veya psikoaktif madde içeren bitkiler ve mantarlar ve adli palinoloji ve mikropların gizemli dünyası: Adli mikrobiyoloji, biyoterör, adli mikrobiyota ve metagenomik analiz uygulamaları ve adli viroloji olmak üzere 3 temel modülde ve toplamda 10 bölümde ele alınmıştır. Kitabın ikici cildinde ise adli bilimlerde biyolojik delil toplama ve incelenme süreçleri: Ölüm zamanı ve çürüme, olay yerlerinden biyolojik delillerin toplanması ve saklanması, cinsel saldırı olgularinda biyolojik örnek alma, adli seroloji ve son olarak da adli genetikte tarihsel gelişim ve güncel uygulamalar: Geçmişten günümüze adli genetikte polimorfizm, adli bilimlerde yeni nesil dizileme, kayıp kişilerin DNA bazlı yöntemlerle kimliklendirilmesi, DNA veri bankaları, adli genetik istatistiği, adli laboratuvarlarda standardizasyon ve akreditasyon, adli mikrobiyolojide metagenomik analiz uygulamaları ve adli genetikte güncel yaklaşımların yer aldığı 2 modül ve 11 bölümden oluşmaktadır. Bu kitap, konusunda uzman olan yazarların deneyimleriyle desteklenmiş hem teorik bilgi hem de pratik uygulamalar hakkında bilgi sunmayı amaçlamaktadır
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Background Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) that reached an epidemic scale in the Middle Ages. Nowadays, the disease is absent in Europe and host genetic influences have been considered as a contributing factor to leprosy disappearance. In this study, a case-control association analysis between multiple human leukocyte antigen (HLA) alleles and leprosy was performed in a medieval European population for the first time. The sample comprised 293 medieval individuals from 18 archaeological sites in Denmark (N = 16) and Germany (N = 2). Results Our results indicate that HLA-B*38 was associated with leprosy risk. Furthermore, we detected three novel variants that were possibly involved in leprosy susceptibility (HLA-A*23, DRB1*13 and DPB1*452). Interestingly, we noted a subtle temporal change in frequency for several alleles previously associated with infectious diseases, inflammatory disorders and cancer in present-day populations. Conclusions This study demonstrates the potential of ancient DNA in the identification of genetic variants involved in predisposition to diseases that are no longer present in Europe but remain endemic elsewhere. Although it is difficult to pinpoint the reason behind the temporal frequency shift, past epidemics of infectious diseases have likely influenced the HLA pool in present-day Europe.
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Several dog skeletons were excavated at the Roman town of Augusta Raurica and at the military camp of Vindonissa, located in the northern Alpine region of Switzerland (Germania Superior). The relationships between them and the people, the nature of their lives, and the circumstances of their deaths are unclear. In order to gain insight into this dog population, we collected 31 dogs deposited almost simultaneously in two wells (second half of the third century CE), three dogs from burial contexts (70–200 CE and third to fifth century CE) at Augusta Raurica, and two dogs from burial contexts at Vindonissa (ca. first century CE). We detected a mixed population of young and adult dogs including small, medium and large sized individuals. Three small dogs had conspicuous phenotypes: abnormally short legs, and one with a brachycephalic skull. Stable isotope analysis of a subset of the dogs showed that their diets were omnivorous with a substantial input of animal proteins and little variation, except one with a particularly low δ ¹⁵ N value, indicating a diet low in animal proteins. Partial mitochondrial DNA sequences from 25 dogs revealed eight haplotypes within canine haplogroup A (11 dogs; 44%; 5 haplotypes), C (8 dogs; 32%; 1 haplotype), D (4 dogs, 16%; 1 haplotype) and B (2 dogs, 8%; 1 haplotype). Based on shotgun sequencing, four Roman mitogenomes were assembled, representing sub-haplogroups A1b3, A1b2 and C2. No canine pathogens were identified, weakening the assumption of infectious disease as a cause for dog disposal. The genetic and morphological diversity observed in dogs of Augusta Raurica and Vindonissa is similar to modern dog diversity.
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The study of well-preserved organic matter (OM) within mineral concretions has provided key insights into depositional and environmental conditions in deep time. Concretions of varied compositions, including carbonate, phosphate, and iron-based minerals, have been found to host exceptionally preserved fossils. Organic geochemical characterization of concretion-encapsulated OM promises valuable new information of fossil preservation, paleoenvironments, and even direct taxonomic information to further illuminate the evolutionary dynamics of our planet and its biota. Full exploitation of this largely untapped geochemical archive, however, requires a sophisticated understanding of the prevalence, formation controls and OM sequestration properties of mineral concretions. Past research has led to the proposal of different models of concretion formation and OM preservation. Nevertheless, the formation mechanisms and controls on OM preservation in concretions remain poorly understood. Here we provide a detailed review of the main types of concretions and formation pathways with a focus on the role of microbes and their metabolic activities. In addition, we provide a comprehensive account of organic geochemical, and complimentary inorganic geochemical, morphological, microbial and paleontological, analytical methods, including recent advancements, relevant to the characterization of concretions and sequestered OM. The application and outcome of several early organic geochemical studies of concretion-impregnated OM are included to demonstrate how this underexploited geo-biological record can provide new insights into the Earth’s evolutionary record. This paper also attempts to shed light on the current status of this research and major challenges that lie ahead in the further application of geo-paleo-microbial and organic geochemical research of concretions and their host fossils. Recent efforts to bridge the knowledge and communication gaps in this multidisciplinary research area are also discussed, with particular emphasis on research with significance for interpreting the molecular record in extraordinarily preserved fossils.
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Vol. 1 Gräber von Pharaonen und Königinnen des alten Ägypten, die noch zu finden sind https://www.amazon.de/Unter-dem-Siegel-Nekropole-K%C3%B6niginnen/dp/3844296395/
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This study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. In addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS™ DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost microbiologists to acquire contaminant-free DNA from permafrost with low biomass. IMPORTANCE Permafrost has the capacity to preserve microbial and non-microbial genomic material for millennia; however, major challenges are associated with permafrost samples, including decontamination of samples and acquiring pure DNA. Contamination of samples during coring and post coring handling and processing could affect downstream analyses and interpretations. Despite the use of multiple different decontamination and DNA extraction methods in studies of permafrost, the efficacy of these methods is not well known. We used a biological tracer to test the efficacy of previously published decontamination methods, as well as a bleach-based method we devised, on two chemically and structurally different permafrost core sections. Our method was the only one that removed all detectable tracer. In addition, we tested multiple DNA extraction kits and modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some extent from peaty, permafrost samples.
Chapter
The rise of more sophisticated forms of analysis has allowed bioarchaeologists to address and answer a wide range of questions regarding past diets, health, mobility, population history, kinship, and taphonomy. However, all of these techniques, e.g. DNA analysis, radiocarbon dating, isotope analysis, and histological analysis require destructive sampling of human remains, which raises ethical issues pertaining to preservation and survival as well as cultural concerns of both past and contemporary societies regarding the post mortem treatment of the dead. This chapter will explore the validity of conducting destructive sampling for the purpose of academic research. It will explore how curators, bioarchaeologists, and archaeologists currently deal with ethical issues surrounding destructive sampling and associated analyses, including the curation of skeletal remains for research purposes, access enquiries, and matters of consent. It is recommended that bioarchaeologists, archaeologists, and curators ensure ethics are at the core of all work carried out when working with human remains. It is thus proposed that these methods should be reserved for focused research questions as opposed to exploratory studies. It is also recommended that researchers and curators receive adequate training in procedures related to destructive sampling as a means of controlling the number of times samples can be taken from bones and teeth which will, in turn, preserve skeletal remains for future generations to study using even more advanced techniques. Following an introduction to the subject matter, this chapter will explore ethics and human remains, technical analyses applied to archaeological human remains, religious and cultural beliefs, and finally makes recommendations for best practice when conducting destructive sampling.
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The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800–1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0–100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract.
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DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.
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Apparently ancient DNA has been reported from amber-preserved insects many millions of years old. Rigorous attempts to reproduce these DNA sequences from amber- and copal-preserved bees and flies have failed to detect any authentic ancient insect DNA. Lack of reproducibility suggests that DNA does not survive over millions of years even in amber, the most promising of fossil environments.
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The expansion of premodern humans into western and eastern Europe approximately 40,000 years before the present led to the eventual replacement of the Neanderthals by modern humans approximately 28,000 years ago. Here we report the second mitochondrial DNA (mtDNA) analysis of a Neanderthal, and the first such analysis on clearly dated Neanderthal remains. The specimen is from one of the eastern-most Neanderthal populations, recovered from Mezmaiskaya Cave in the northern Caucasus. Radiocarbon dating estimated the specimen to be approximately 29,000 years old and therefore from one of the latest living Neanderthals. The sequence shows 3.48% divergence from the Feldhofer Neanderthal. Phylogenetic analysis places the two Neanderthals from the Caucasus and western Germany together in a clade that is distinct from modern humans, suggesting that their mtDNA types have not contributed to the modern human mtDNA pool. Comparison with modern populations provides no evidence for the multiregional hypothesis of modern human evolution.
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At the recent 5th International Ancient DNA Conference in Manchester, U.K., reported by Erik Stokstad in his News Focus article “Divining diet and disease from DNA” (28 Jul., p. [530][1]), one presentation boldly opened with the claim that the field was now mature and could move ahead with
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Domestication entails control of wild species and is generally regarded as a complex process confined to a restricted area and culture. Previous DNA sequence analyses of several domestic species have suggested only a limited number of origination events. We analyzed mitochondrial DNA (mtDNA) control region sequences of 191 domestic horses and found a high diversity of matrilines. Sequence analysis of equids from archaeological sites and late Pleistocene deposits showed that this diversity was not due to an accelerated mutation rate or an ancient domestication event. Consequently, high mtDNA sequence diversity of horses implies an unprecedented and widespread integration of matrilines and an extensive utilization and taming of wild horses. However, genetic variation at nuclear markers is partitioned among horse breeds and may reflect sex-biased dispersal and breeding.
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DNA was extracted from three fecal samples, more than 2,000 years old, from Hinds Cave, Texas. Amplification of human mtDNA sequences showed their affiliation with contemporary Native Americans, while sequences from pronghorn antelope, bighorn sheep, and cottontail rabbit allowed these animals to be identified as part of the diet of these individuals. Furthermore, amplification of chloroplast DNA sequences identified eight different plants as dietary elements. These archaic humans consumed 2-4 different animal species and 4-8 different plant species during a short time period. The success rate for retrieval of DNA from paleofeces is in strong contrast to that from skeletal remains where the success rate is generally low. Thus, human paleofecal remains represent a source of ancient DNA that significantly complements and may in some cases be superior to that from skeletal tissue.
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Anthropologists were quick to recognize the potential of new techniques in molecular biology to provide additional lines of evidence on questions long investigated in anthropology, as well as those questions that, while always of interest, could not have been addressed by more traditional techniques. The earliest ancient DNA studies, both within anthropology and in other fields, lacked rigorous hypothesis testing. However, more recently the true value of ancient DNA studies is being realized, and methods are being applied to a wide variety of anthropological questions. We review the most common methods and applications to date, and describe promising avenues of future research. We find that ancient DNA analyses have a valuable place in the array of anthropological techniques, but argue that such studies must not be undertaken merely to demonstrate that surviving DNA is present in organic remains, and that no such work should be performed before a careful consideration of the possible ethical ramifications of the research is undertaken.
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Genetic analyses of permafrost and temperate sediments reveal that plant and animal DNA may be preserved for long periods, even in the absence of obvious macrofossils. In Siberia, five permafrost cores ranging from 400,000 to 10,000 years old contained at least 19 different plant taxa, including the oldest authenticated ancient DNA sequences known, and megafaunal sequences including mammoth, bison, and horse. The genetic data record a number of dramatic changes in the taxonomic diversity and composition of Beringian vegetation and fauna. Temperate cave sediments in New Zealand also yielded DNA sequences of extinct biota, including two species of ratite moa, and 29 plant taxa characteristic of the prehuman environment. Therefore, many sedimentary deposits may contain unique, and widespread, genetic records of paleoenvironments.
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When highly efficient polymerase was used with high cycle numbers (50-60), strong amplifications were observed, but negative controls were also unexpectedly amplified in a study of ancient human mtDNA from 2000-year-old skeletons. The results of a series of tests revealed that the hypersensitive polymerase chain reaction (PCR) generated by higher cycles and the presence of contaminant DNA (though at extremely low levels) should be responsible for the amplification of negative controls. We suggest that PCR sensitivity be optimized to take advantage of highly efficient polymerase and at the same time prevent "background DNA" from becoming "contaminant DNA" and obscuring the analysis of authentic ancient DNA. We propose the use of multiple positive controls when amplifying ancient human mtDNA samples to indicate the sensitivity of individual PCR amplifications and to monitor the contamination levels of modern human DNA. This study provides some suggestions as to how to amplify and analyze ancient human mtDNA when unavoidable and extremely tiny amounts of modern human DNA exist.
Article
Using polymerase chain reaction (PCR) amplification, it is possible to analyze DNA from limited source template. This method has proved especially valuable in studies of ancient DNA and in forensic investigations. However, PCR reactions containing minimal or damaged source template are prone to contamination by DNA from a number of other sources. While standard protocols to prevent and/or detect contamination do exist, methods of eliminating contamination are needed to ensure the validity of results obtained. We present a method to eliminate sources of contamination in reagents and labware through the use of a DNase prior to PCR amplification without damaging even the minimal amounts of template present in ancient DNA samples. This method, suggested previously for forensics applications, appears to be effective in eliminating contamination without interfering with the amplification of ancient template.
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A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
Article
Apparently ancient DNA has been reported from amber-preserved insects many millions of years old. Rigorous attempts to reproduce these DNA sequences from amber- and copal-preserved bees and flies have failed to detect any authentic ancient insect DNA. Lack of reproducibility suggests that DNA does not survive over millions of years even in amber, the most promising of fossil environments.
Article
Out of molecular biology – the revolution of past decades in biological science – there now begins to come molecular archaeology, the study of DNA in ancient plants, animals and people to address questions of history as well as biology. Here is set out what molecular archaeology is about, how it works and what it has begun to do.
Article
A polymerase chain reaction (PCR) method has been used to confirm the presence of tuberculosis bacterial DNA in extracts of three human bone specimens with lesions suggestive of this disease. A fused wrist bone and a lumbar vertebra from the mediaeval period were found to be positive and an adjacent lumbar vertebra was weakly positive. A control vertebra from the same period (1350–1538 ï¿¿ï¿¿) from an individual with bone lesions characteristic of syphilis was negative. Against a background of partially degraded DNA, nested PCR was found to be superior for detecting tuberculosis bacterial sequences compared with reamplification of first-round products or modification of other PCR stages. DNA survival in bone was assessed using PCR for a human-specific region of the Alu repetitive short interspersed nucleotide elements (SINES) and the ability to amplify a single copy gene. Genomic DNA was found in all bones examined but the single copy gene did not amplify from the vertebra weakly positive for tuberculosis or the control. This may represent relatively poor DNA preservation in these samples. These findings corroborate the use of PCR as an aid in the study of infectious disease in ancient bone samples and stress the importance of assessing native DNA survival.
Data
A polymerase chain reaction (PCR) method has been used to confirm the presence of tuberculosis bacterial DNA in extracts of three human bone specimens with lesions suggestive of this disease. A fused wrist bone and a lumbar vertebra from the mediaeval period were found to be positive and an adjacent lumbar vertebra was weakly positive. A control vertebra from the same period (1350–1538 ï¿¿ï¿¿) from an individual with bone lesions characteristic of syphilis was negative. Against a background of partially degraded DNA, nested PCR was found to be superior for detecting tuberculosis bacterial sequences compared with reamplification of first-round products or modification of other PCR stages. DNA survival in bone was assessed using PCR for a human-specific region of the Alu repetitive short interspersed nucleotide elements (SINES) and the ability to amplify a single copy gene. Genomic DNA was found in all bones examined but the single copy gene did not amplify from the vertebra weakly positive for tuberculosis or the control. This may represent relatively poor DNA preservation in these samples. These findings corroborate the use of PCR as an aid in the study of infectious disease in ancient bone samples and stress the importance of assessing native DNA survival.
Article
The survival of DNA, the most informative biological molecule, for periods of at least several thousand years in bone was demonstrated more than four years ago. However, difficulties with authenticating ancient DNA have made diagenetic studies problematic. It is therefore essential that these problems be overcome before the question of DNA survival can be addressed. Here we describe our work with a range of Holocene skeletal material from domestic animals and humans, and discuss how we go about authenticating the results. In the first instance, results should be reproducible between different laboratories, in order to eliminate the possibility of laboratory-specific contamination. The main risk is then contamination of the material prior to sampling for DNA. It is therefore important to have criteria whereby the authenticity of the results can be evaluated. These include amplification with species-specific primers to target DNA from domestic animals, sex determination in humans, and phylogenetic position in both. Since animals can be tested for the presence of contaminating human DNA, work with animals can be used to control and assess methodology. Our initial studies in this area suggest that more than 50% of skeletal remains from the past two thousand years are likely to contain amplifiable endogenous DNA, but that in the case of human material great care is needed to distinguish this from contamination introduced before the samples reach the laboratory.
Article
Nucleic acids are preserved in prehistoric samples under a wide range of depositional environments. The development of new molecular methods, especially the polymerase chain reaction, has made possible the recovery and manipulation of these molecules, and the subsequent molecular genetic characterization of the ancient samples. The analysis of ancient (a)DNA is complicated by the degraded nature of ancient nucleic acids, as well as the presence of enzymatic inhibitors in aDNA extracts. We review aspects of ancient DNA preservation, a variety of methods for the extraction and amplification of informative DNA segments from ancient samples, and the diffi-culties encountered in documenting the authenticity of ancient DNA template. Studies using aDNA to address questions in human population history or human evolution are reviewed and discussed. Future prospects for the field and potential directions for future aDNA research efforts in physical anthropology are identified.
Article
A decade ago, the first reviews of the collective mitochondrial DNA (mtDNA) data from Native Americans concluded that the Americas were peopled through multiple migrations from different Asian populations beginning more than 30,000 years ago.1 These reports confirmed multiple-wave hypotheses suggested earlier by other sources and rejected the dominant Clovis-first archeological paradigm. Consequently, it appeared that molecular biology had made a significant contribution to the study of American prehistory. As Cann2 comments, the Americas held the greatest promise for genetics to help solve some of the mysteries of prehistoric populations. In particular, mtDNA appeared to offer real potential as a means of better understanding ancient population movements. A decade later, none of the early conclusions remain unequivocal. Nevertheless, in its maturity, the study of Native American mtDNA has produced a volume of reports that still illuminate the nature and timing of the first peopling and postcolonization population movements within the New World.
Article
The presence of DNA has been demonstrated in the cell nuclei of an ancient Egyptian mummy fragment. When extracted, this DNA proved to be degraded to a considerable extent and chemically modified. However, the preservation of nucleic acids in this specimen suggests that applying recombinant DNA techniques to the study of ancient mummified tissues might prove to be a fruitful future area of research.
Article
This paper reports on the development and application of methods for using DNA analysis for species identification of archaeological salmon bone. Short fragments (less than 300 bp) of mitochondrial DNA from the control region (D-loop) and cytochrome B (CytB) gene were targeted for amplification using the polymerase chain reaction (PCR) technique. The method was used on more than 20 salmon bone samples (dated 7000 to 2000 BP) from the site of Namu on the central coast of British Columbia. Four species: coho, sockeye, pink and chum salmon were identified from the samples. The results are considered valid since systematic contaminations were not detected, multiple species and multiple DNA haplotypes of the same species were identified from the same set of bone samples, and the identified species are consistent with those inferred from other lines of evidence. The results demonstrate the applicability of the ancient DNA technique to species identification of even single salmon vertebrae from archaeological sites in the Pacific Northwest of North America.
Article
One of the problems faced by archaeologists is in identifying species from fragmented bone recovered in sites. We felt that PCR/DNA technology offered the means by which species could be identified from such remains. We used fragmented bones from the site of Head-Smashed-In Buffalo Jump to establish protocols for PCR/DNA analysis. Fragmented bones from sites in Northern Nigeria and Northern Cameroon were then analysed. Positive identification of sheep and goat were made on five of the 14 bones tested.
Article
Thousands of Late Pleistocene remains are found in sites throughout Beringia. These specimens comprise an Ice Age genetic museum, and the DNA contained within them provide a means to observe evolutionary processes within populations over geologically significant time scales. Phylogenetic analyses can identify the taxonomic positions of extinct species and provide estimates of speciation dates. Geographic and temporal divisions apparent in the genetic data can be related to ecological change, human impacts, and possible landscape mosaics in Beringia. The application of ancient DNA techniques to traditional paleontological studies provides a new perspective to long-standing questions regarding the paleoenvironment and diversity of Late Pleistocene Beringia.
Article
Ancient DNA research from its inception in the 1980s is reviewed in the context of pre-Columbian archaeology. Changing approaches to the recovery of DNA from human remains are followed, and the accumulated records considered in the light of hypotheses about the post-Columbian depletion and augmentation of the New World gene pool.
Article
DNA was extracted from the Neandertal-type specimen found in 1856 in western Germany. By sequencing clones from short overlapping PCR products, a hitherto unknown mitochondrial (mt) DNA sequence was determined. Multiple controls indicate that this sequence is endogenous to the fossil. Sequence comparisons with human mtDNA sequences, as well as phylogenetic analyses, show that the Neandertal sequence falls outside the variation of modern humans. Furthermore, the age of the common ancestor of the Neandertal and modern human mtDNAs is estimated to be four times greater than that of the common ancestor of human mtDNAs. This suggests that Neandertals went extinct without contributing mtDNA to modern humans.
Article
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
Article
Pieces of mitochondrial DNA from a 7000-year-old human brain were amplified by the polymerase chain reaction and sequenced. Albumin and high concentrations of polymerase were required to overcome a factor in the brain extract that inhibits amplification. For this and other sources of ancient DNA, we find an extreme inverse dependence of the amplification efficiency on the length of the sequence to be amplified. This property of ancient DNA distinguishes it from modern DNA and thus provides a new criterion of authenticity for use in research on ancient DNA. The brain is from an individual recently excavated from Little Salt Spring in southwestern Florida and the anthropologically informative sequences it yielded are the first obtained from archaeologically retrieved remains. The sequences show that this ancient individual belonged to a mitochondrial lineage that is rare in the Old World and not previously known to exist among Native Americans. Our finding brings to three the number of maternal lineages known to have been involved in the prehistoric colonization of the New World.
Article
The study of ancient DNA offers the possibility of following genetic change over time. However, the field is plagued by a problem which is unique in molecular biology--the difficulty of verifying results by reproduction. Some of the reasons for this are technical and derive from the low copy number and damaged state of ancient DNA molecules. Other reasons are the unique nature of many of the objects from which DNA is extracted. We describe methodological approaches with which these problems can be alleviated in order to ensure that results are scientific in the sense that they can be reproduced by others.
Article
Ancient DNA was obtained from skeletal remains from the Norris Farms #36 cemetery, a pre-Columbian archeological site in central Illinois that dates to A.D. 1300. Four mitochondrial DNA (mtDNA) markers were analyzed that delineate the four primary mtDNA lineages found in contemporary Amerindian populations. mtDNA types were determined for 50 individuals; 49 belonged to one of these four lineages. One lineage occurred only in males, suggesting an immigration of maternally related males into this community. There was no significant spatial patterning of mtDNA lineages within the cemetery. This survey of ancient DNA variation in a pre-Columbian population supports the view that the initial colonization of the New World comprised just four primary mtDNA lineages.
Article
Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.
Article
Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.