Historical Collections of Tropical Marine Mammals Are an Excellent Resource for Ancient DNA.

The field of ancient DNA is dominated by studies focusing on terrestrial vertebrates. This taxonomic bias limits our understanding of endogenous DNA preservation for species with different bone physiology, such as teleost fish. Teleost bone is typically brittle, porous, lightweight, and is characterized by a lack of bone remodeling during growth. All of these factors potentially affect DNA preservation. Using high-throughput shotgun sequencing, we here investigate the preservation of DNA in a range of different bone elements from over 200 archaeological Atlantic cod (Gadus morhua) specimens from 38 sites in northern Europe, dating up to 8000 years before present. We observe that the majority of archaeological sites (79%) yield endogenous DNA, with 40% of sites providing samples containing high levels (>20%). Library preparation success and levels of endogenous DNA depend mainly on excavation site and pre-extraction laboratory treatment. The use of pre-extraction treatments lowers the rate of libraries that can be sequenced, although — if successful — the fraction of endogenous DNA can be improved by several orders of magnitude. This trade-off between library preparation success and levels of endogenous DNA allows for alternative extraction strategies depending on the requirements of down-stream analyses and research questions. Finally, we do not find particular bone elements to yield higher levels of endogenous DNA, as is the case for denser bones in mammals. Our results highlight the potential of archaeological fish bone as a source for ancient DNA and suggest a possible role of bone remodeling in the preservation of endogenous DNA.

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.

Molecular biology has made considerable contributions to the study of human history from the earliest days of our species, when our ancestors began to walk on the savannah, to the dawn of civilization and the more recent history of mankind. The analysis of contemporary mitochondrial DNA, for example, has told us that Homo sapiens first appeared in Africa; the sequencing of the Neanderthal genome has revealed that Homo sapiens and Homo neanderthalensis lived along each other and interbred before the latter disappeared; last year, the analysis of DNA extracted from a skeleton found under a parking lot in Leicester, UK, showed that it was the remains of King Richard III. Although the advent of next‐generation sequencing and advances in bioinformatics have boosted research using ancient DNA, a major hurdle has always been the quantity and quality of the DNA itself: DNA molecules quickly degrade over time into smaller fragments, and microbial contamination makes it challenging to identify human fragments within a sample. Intriguingly, teeth have proven to be an excellent source of high‐quality ancient DNA, yielding insights into the evolution of the human diet, disease and immunity since the onset of agriculture some 10,000 years ago. Dental calculus has been the most complete source of historical sequence data, especially for probing oral microbiome populations. At the same time, dental pulp—the connective tissue at the centre of teeth—has been an important source of information about ancient diseases. Archaeologist Keith Dobney, from the University of Aberdeen in the UK, and his colleagues first identified the potential of dental calculus as a source of information about past diets and microbial populations long before the advent of ancient DNA studies [1]. Now, almost 30 years later, Dobney is among those at the forefront of the revolution in ancient DNA analysis. “It appears that dental …

Advances in the field of museomics have promoted a high sampling demand for natural history collections (NHCs), eventually resulting in damage to invaluable resources to understand historical biodiversity. It is thus essential to achieve a consensus about which historical tissues present the best sources of DNA. In this study, we evaluated the performance of different historical tissues from Iberian wolf NHCs in genome-wide assessments. We targeted three tissues—bone (jaw and femur), maxilloturbinal bone, and skin—that have been favored by traditional taxidermy practices for mammalian carnivores. Specifically, we performed shotgun sequencing and target capture enrichment for 100,000 single nucleotide polymorphisms (SNPs) selected from the commercial Canine HD BeadChip across 103 specimens from 1912 to 2005. The performance of the different tissues was assessed using metrics based on endogenous DNA content, uniquely high-quality mapped reads after capture, and enrichment proportions. All samples succeeded as DNA sources, regardless of their collection year or sample type. Skin samples yielded significantly higher amounts of endogenous DNA compared to both bone types, which yielded equivalent amounts. There was no evidence for a direct effect of tissue type on capture efficiency; however, the number of genotyped SNPs was strictly associated with the starting amount of endogenous DNA. Evaluation of genotyping accuracy for distinct minimum read depths across tissue types showed a consistent overall low genotyping error rate (<7%), even at low (3x) coverage. We recommend the use of skins as reliable and minimally destructive sources of endogenous DNA for whole-genome and target enrichment approaches in mammalian carnivores. In addition, we provide a new 100,000 SNP capture array validated for historical DNA (hDNA) compatible to the Canine HD BeadChip for high-quality DNA. The increasing demand for NHCs as DNA sources should encourage the generation of genomic datasets comparable among studies.

Ancient DNA research has developed rapidly over the past few decades due to the improvement in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from the raw sequencing data. This is often difficult due to the degradation of ancient DNA and high levels of contamination, especially homologous contamination. In this study, we collected whole genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from the homologous contaminations, we attempted to recover reads based on the ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing the homologous contaminations to a very low proportion. Overall, these recommendations for ancient DNA mapping and separation of endogenous DNA in this study could facilitate future studies of ancient DNA.

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Mammalian mitochondrial capture, a tool for rapid screening of DNA preservation in faunal and undiagnostic remains, and its application to Middle Pleistocene specimens from Qesem Cave (Israel)

Density separation is a process routinely used to segregate minerals, organic matter, and even microplastics, from soils and sediments. Here we apply density separation to archaeological bone powders before DNA extraction to increase endogenous DNA recovery relative to a standard control extraction of the same powders. Using nontoxic heavy liquid solutions, we separated powders from the petrous bones of 10 individuals of similar archaeological preservation into eight density intervals (2.15 to 2.45 g/cm3, in 0.05 increments). We found that the 2.30 to 2.35 g/cm3 and 2.35 to 2.40 g/cm3 intervals yielded up to 5.28-fold more endogenous unique DNA than the corresponding standard extraction (and up to 8.53-fold before duplicate read removal), while maintaining signals of ancient DNA authenticity and not reducing library complexity. Although small 0.05 g/cm3 intervals may maximally optimize yields, a single separation to remove materials with a density above 2.40 g/cm3 yielded up to 2.57-fold more endogenous DNA on average, which enables the simultaneous separation of samples that vary in preservation or in the type of material analyzed. While requiring no new ancient DNA laboratory equipment and fewer than 30 min of extra laboratory work, the implementation of density separation before DNA extraction can substantially boost endogenous DNA yields without decreasing library complexity. Although subsequent studies are required, we present theoretical and practical foundations that may prove useful when applied to other ancient DNA substrates such as teeth, other bones, and sediments.

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. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA. Am J Phys Anthropol 111:5–23, 2000. © 2000 Wiley-Liss, Inc.

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. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA.

Ancient DNA diffuses from human bones to cave stones

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.

Paleomicrobiology is a special branch of micropaleontology concerned with the study of bacterial fossils. We have used the term 'oral paleomicrobiology', as in this review we have focused on the ancient oral microflora. Recently, dental calculus and dental pulp have been identified as rich sources of ancient microbial DNA. Study of this ancient genetic material opens a new door to the ancient world. This review gives an overview of history of ancient DNA research, various techniques of analyzing ancient DNA in dental calculus and dental pulp, and the implications of the oral paleomicrobiology. A comprehensive literature search was performed in the following databases-pubmed, medline and google scholar for studies published before 10 April, 2015. The following keywords were used- 'ancient DNA', 'ancient oral flora, 'oral paleomicrobiology' and 'oral microbiome', '16S rRNA sequencing'. To obtain additional data, a manual search was performed using the reference lists of selected articles. As a result of literature search, 27 articles were found in pubmed, 12 in google scholar and one in medline. Eight more articles were selected from the reference list of selected articles. The combination of microbiology and paleontology has brought a revolution in the study of human evolution and microbial communities. The naturally well-preserved samples of microbial DNA from dental pulp and microbial colonies trapped in dental calculus are a potential source of microbial genetic material, which will prove invaluable in resolving mysteries of the past. This may be a beginning of a new era of oral paleomicrobiology, which will contribute in our studies about prevention of disease by establishing symbiosis between human beings and their microbiome.

High-throughput sequencing methods have facilitated obtaining large amounts of data from degraded DNA, thus resulting in a dramatic increase in destructive sampling requests to museums. Because the tissues taken from museum specimens as sources of DNA are destroyed during analysis, consideration of the costs and benefits of loss of valuable specimen material relative to knowledge gained is required for any project utilizing destructive sampling. Variation exists in the preservation of DNA in historical specimens due to specimen age and type of museum preparation, among other factors. Thus, it is important to assess DNA yield and quality from different sources of museum specimens when considering the needs of a particular molecular project. We compared DNA derived from several common sources of museum specimens including bone, claw, skin, and soft tissue adherent to skeletal preparations. To account for differences in preparation type and therefore specimen preservation, we tested the performance of samples representing 3 taxonomic groups: mephitids, rodents, and marsupials. We also compared yields from 2 commonly used DNA extraction techniques. DNA quality was assessed by comparing average fragment size, concentration, and copy number of template DNA (for mitochondrial and nuclear markers) in genomic DNA extracts, as well as mitochondrial genome sequence coverage resulting from shotgun sequencing. We show that DNA quality derived from historic museum samples differs depending on specimen and sample type; however, all samples yielded high mitochondrial copy number except the skin and nail from the tanned specimen. Overall, claw samples produced the greatest number of high-quality sequencing reads with the least amount of bacterial contamination. We also found that high DNA concentrations did not necessarily result in high percentages of on-target reads; in fact, the samples that yielded the highest DNA quantities also had the highest amount of exogenous bacterial DNA. Our results indicate that most historical tissue types can be suitable for next-generation sequencing approaches, therefore providing multiple options for natural history collection staff and researchers when considering destructive sampling requests.

Extracellular DNA is ubiquitous in soil and sediment and constitutes a dominant fraction of environmental DNA in aquatic systems. In theory, extracellular DNA is composed of genomic elements persisting at different degrees of preservation produced by processes occurring on land, in the water column and sediment. Extracellular DNA can be taken up as a nutrient source, excreted or degraded by microorganisms, or adsorbed onto mineral matrices, thus potentially preserving information from past environments. To test whether extracellular DNA records lacustrine conditions, we sequentially extracted extracellular and intracellular DNA from anoxic sediments of ferruginous Lake Towuti, Indonesia. We applied 16S rRNA gene Illumina sequencing on both fractions to discriminate exogenous from endogenous sources of extracellular DNA in the sediment. Environmental sequences exclusively found as extracellular DNA in the sediment originated from multiple sources. For instance, Actinobacteria, Verrucomicrobia, and Acidobacteria derived from soils in the catchment. Limited primary productivity in the water column resulted in few sequences of Cyanobacteria in the oxic photic zone, whereas stratification of the water body mainly led to secondary production by aerobic and anaerobic heterotrophs. Chloroflexi and Planctomycetes, the main degraders of sinking organic matter and planktonic sequences at the water-sediment interface, were preferentially preserved during the initial phase of burial. To trace endogenous sources of extracellular DNA, we used relative abundances of taxa in the intracellular DNA to define which microbial populations grow, decline or persist at low density with sediment depth. Cell lysis became an important additional source of extracellular DNA, gradually covering previous genetic assemblages as other microbial genera became more abundant with depth. The use of extracellular DNA as nutrient by active microorganisms led to selective removal of sequences with lowest GC contents. We conclude that extracellular DNA preserved in shallow lacustrine sediments reflects the initial environmental context, but is gradually modified and thereby shifts from its stratigraphic context. Discrimination of exogenous and endogenous sources of extracellular DNA allows simultaneously addressing in-lake and post-depositional processes. In deeper sediments, the accumulation of resting stages and sequences from cell lysis would require stringent extraction and specific primers if ancient DNA is targeted.