Ancient DNA SNP-panel data suggests stability in bluefin tuna genetic diversity despite centuries of fluctuating catches in the eastern Atlantic and Mediterranean.

Atlantic bluefin tuna (Thunnus thynnus; BFT) abundance was depleted in the late 20th and early 21st century due to overfishing. Historical catch records further indicate that the abundance of BFT in the Mediterranean has been fluctuating since at least the 16th century. Here we build upon previous work on ancient DNA of BFT in the Mediterranean by comparing contemporary (2009-2012) specimens with archival (1911-1926) and archaeological (2nd century BCE-15th century CE) specimens that represent population states prior to these two major periods of exploitation, respectively. We successfully genotyped and analysed 259 contemporary and 123 historical (91 archival and 32 archaeological) specimens at 92 SNP loci that were selected for their ability to differentiate contemporary populations or their association with core biological functions. We found no evidence of genetic bottlenecks, inbreeding or population restructuring between temporal sample groups that might explain what has driven catch fluctuations since the 16th century. We also detected a putative adaptive response, involving the cytoskeletal protein synemin which may be related to muscle stress. However, these results require further investigation with more extensive genome-wide data to rule out demographic changes due to overfishing, and other natural and anthropogenic factors, in addition to elucidating the adaptive drivers related to these.

Chronic active non-lethal human-type tuberculosis in a high royal Bavarian officer of Napoleonic times-a mummy study.

In paleopathology, morphological and molecular evidence for infection by mycobacteria of the M. tuberculosis complex (MTC) is frequently associated with early death. In the present report, we describe a multidisciplinary study of a well-preserved mummy from Napoleonic times with a long-standing tuberculous infection by M. tuberculosis senso stricto who died at the age of 88 years of focal and non-MTB related bronchopneumonia. The well-preserved natural mummy of the Royal Bavarian General, Count Heinrich LII Reuss-Köstritz (1763-1851 CE), was extensively investigated by macro- and histomorphology, whole body CT scans and organ radiography, various molecular tissue analyses, including stable isotope analysis and molecular genetic tests. We identified signs for a long-standing, but terminally inactive pulmonary tuberculosis, tuberculous destruction of the second lumbar vertebral body, and a large tuberculous abscess in the right (retroperitoneal) psoas region (a cold abscess). This cold abscess harboured an active tuberculous infection as evidenced by histological and molecular tests. Radiological and histological analysis further revealed extensive arteriosclerosis with (non-obliterating) coronary and significant carotid arteriosclerosis, healthy bone tissue without evidence of age-related osteopenia, evidence for diffuse idiopathic skeletal hyperostosis and mild osteoarthrosis of few joints. This suggests excellent living conditions correlating well with his diet indicated by stable isotope results and literary evidence. Despite the clear evidence of a tuberculous cold abscess with bacterioscopic and molecular proof for a persisting MTC infection of a human-type M. tuberculosis strain, we can exclude the chronic MTC infection as cause of death. The detection of MTC in historic individuals should therefore be interpreted with great caution and include further data, such as their nutritional status.

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA.

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

Systematic benchmark of ancient DNA read mapping.

The current standard practice for assembling individual genomes involves mapping millions of short DNA sequences (also known as DNA 'reads') against a pre-constructed reference genome. Mapping vast amounts of short reads in a timely manner is a computationally challenging task that inevitably produces artefacts, including biases against alleles not found in the reference genome. This reference bias and other mapping artefacts are expected to be exacerbated in ancient DNA (aDNA) studies, which rely on the analysis of low quantities of damaged and very short DNA fragments (~30-80 bp). Nevertheless, the current gold-standard mapping strategies for aDNA studies have effectively remained unchanged for nearly a decade, during which time new software has emerged. In this study, we used simulated aDNA reads from three different human populations to benchmark the performance of 30 distinct mapping strategies implemented across four different read mapping software-BWA-aln, BWA-mem, NovoAlign and Bowtie2-and quantified the impact of reference bias in downstream population genetic analyses. We show that specific NovoAlign, BWA-aln and BWA-mem parameterizations achieve high mapping precision with low levels of reference bias, particularly after filtering out reads with low mapping qualities. However, unbiased NovoAlign results required the use of an IUPAC reference genome. While relevant only to aDNA projects where reference population data are available, the benefit of using an IUPAC reference demonstrates the value of incorporating population genetic information into the aDNA mapping process, echoing recent results based on graph genome representations.

Bile acids and oxo-metabolites as markers of human faecal input in the ancient Pompeii ruins.

Small organic molecules, lipids, proteins, and DNA fragments can remain stable over centuries. Powerful and sensitive chemical analysis can therefore be used to characterize ancient remains for classical archaeological studies. This bio-ecological dimension of archaeology can contribute knowledge about several aspects of ancient life, including social organization, daily habits, nutrition, and food storage. Faecal remains (i.e. coprolites) are particularly interesting in this regard, with scientists seeking to identify new faecal markers. Here, we report the analysis of faecal samples from modern-day humans and faecal samples from a discharge pit on the site of the ruins of ancient Pompeii. We propose that bile acids and their gut microbiota oxo-metabolites are the most specific steroid markers for detecting faecal inputs. This is due to their extreme chemical stability and their exclusive occurrence in vertebrate faeces, compared to other ubiquitous sterols and steroids.

A systematic investigation of human DNA preservation in medieval skeletons.

Ancient DNA (aDNA) analyses necessitate the destructive sampling of archaeological material. Currently, the cochlea, part of the osseous inner ear located inside the petrous pyramid, is the most sought after skeletal element for molecular analyses of ancient humans as it has been shown to yield high amounts of endogenous DNA. However, destructive sampling of the petrous pyramid may not always be possible, particularly in cases where preservation of skeletal morphology is of top priority. To investigate alternatives, we present a survey of human aDNA preservation for each of ten skeletal elements in a skeletal collection from Medieval Germany. Through comparison of human DNA content and quality we confirm best performance of the petrous pyramid and identify seven additional sampling locations across four skeletal elements that yield adequate aDNA for most applications in human palaeogenetics. Our study provides a better perspective on DNA preservation across the human skeleton and takes a further step toward the more responsible use of ancient materials in human aDNA studies.

DNA from resin-embedded organisms: Past, present and future.

Past claims have been made for fossil DNA recovery from various organisms (bacteria, plants, insects and mammals, including humans) dating back in time from thousands to several million years BP. However, many of these recoveries, especially those described from million-year-old amber (fossil resin), have faced criticism as being the result of modern environmental contamination and for lack of reproducibility. Using modern genomic techniques, DNA can be obtained with confidence from a variety of substrates (e.g. bones, teeth, gum, museum specimens and fossil insects) of different ages, albeit always less than one million years BP, and results can also be obtained from much older materials using palaeoproteomics. Nevertheless, new attempts to determine if ancient DNA (aDNA) is present in insects preserved in 40 000-year old sub-fossilised resin, the precursor of amber, have been unsuccessful or not well documented. Resin-embedded specimens are therefore regarded as unsuitable for genetic studies. However, we demonstrate here, for the first time, that although a labile molecule, DNA is still present in platypodine beetles (Coleoptera: Curculionidae) embedded in six-year-old and two-year-old resin pieces from Hymenaea verrucosa (Angiospermae: Fabaceae) collected in Madagascar. We describe an optimised method which meets all the requirements and precautions for aDNA experiments for our purpose: to explore the DNA preservation limits in resin. Our objective is far from starting an uncontrolled search for aDNA in amber as it was in the past, but to start resolving basic aspects from the DNA preservation in resin and search from the most modern samples to the ancient ones, step by step. We conclude that it is therefore possible to study genomics from resin-embedded organisms, although the time limits remain to be determined.

Reconstructing double-stranded DNA fragments on a single-molecule level reveals patterns of degradation in ancient samples.

Extensive manipulations involved in the preparation of DNA samples for sequencing have hitherto made it impossible to determine the precise structure of double-stranded DNA fragments being sequenced, such as the presence of blunt ends, single-stranded overhangs, or single-strand breaks. We here describe MatchSeq, a method that combines single-stranded DNA library preparation from diluted DNA samples with computational sequence matching, allowing the reconstruction of double-stranded DNA fragments on a single-molecule level. The application of MatchSeq to Neanderthal DNA, a particularly complex source of degraded DNA, reveals that 1- or 2-nt overhangs and blunt ends dominate the ends of ancient DNA molecules and that short gaps exist, which are predominantly caused by the loss of individual purines. We further show that deamination of cytosine to uracil occurs in both single- and double-stranded contexts close to the ends of molecules, and that single-stranded parts of DNA fragments are enriched in pyrimidines. MatchSeq provides unprecedented resolution for interrogating the structures of fragmented double-stranded DNA and can be applied to fragmented double-stranded DNA isolated from any biological source. The method relies on well-established laboratory techniques and can easily be integrated into routine data generation. This possibility is shown by the successful reconstruction of double-stranded DNA fragments from previously published single-stranded sequence data, allowing a more comprehensive characterization of the biochemical properties not only of ancient DNA but also of cell-free DNA from human blood plasma, a clinically relevant marker for the diagnosis and monitoring of disease.

Integration of ancient DNA with transdisciplinary dataset finds strong support for Inca resettlement in the south Peruvian coast.

Ancient DNA (aDNA) analysis provides a powerful means of investigating human migration, social organization, and a plethora of other crucial questions about humanity's past. Recently, specialists have suggested that the ideal research design involving aDNA would include multiple independent lines of evidence. In this paper, we adopt a transdisciplinary approach integrating aDNA with archaeological, biogeochemical, and historical data to investigate six individuals found in two cemeteries that date to the Late Horizon (1400 to 1532 CE) and Colonial (1532 to 1825 CE) periods in the Chincha Valley of southern Peru. Genomic analyses indicate that these individuals are genetically most similar to ancient and present-day populations from the north Peruvian coast located several hundred kilometers away. These genomic data are consistent with 16th century written records as well as ceramic, textile, and isotopic data. These results provide some of the strongest evidence yet of state-sponsored resettlement in the pre-Colonial Andes. This study highlights the power of transdisciplinary research designs when using aDNA data and sets a methodological standard for investigating ancient mobility in complex societies.

Screening archaeological bone for palaeogenetic and palaeoproteomic studies.

The recovery and analysis of ancient DNA and protein from archaeological bone is time-consuming and expensive to carry out, while it involves the partial or complete destruction of valuable or rare specimens. The fields of palaeogenetic and palaeoproteomic research would benefit greatly from techniques that can assess the molecular quality prior to sampling. To be relevant, such screening methods should be effective, minimally-destructive, and rapid. This study reports results based on spectroscopic (Fourier-transform infrared spectroscopy in attenuated total reflectance [FTIR-ATR]; n = 266), palaeoproteomic (collagen content; n = 226), and palaeogenetic (endogenous DNA content; n = 88) techniques. We establish thresholds for three different FTIR indices, a) the infrared splitting factor [IRSF] that assesses relative changes in bioapatite crystals' size and homogeneity; b) the carbonate-to-phosphate [C/P] ratio as a relative measure of carbonate content in bioapatite crystals; and c) the amide-to-phosphate ratio [Am/P] for assessing the relative organic content preserved in bone. These thresholds are both extremely reliable and easy to apply for the successful and rapid distinction between well- and poorly-preserved specimens. This is a milestone for choosing appropriate samples prior to genomic and collagen analyses, with important implications for biomolecular archaeology and palaeontology.

Mining ancient microbiomes using selective enrichment of damaged DNA molecules.

BACKGROUND: The identification of bona fide microbial taxa in microbiomes derived from ancient and historical samples is complicated by the unavoidable mixture between DNA from ante- and post-mortem microbial colonizers. One possibility to distinguish between these sources of microbial DNA is querying for the presence of age-associated degradation patterns typical of ancient DNA (aDNA). The presence of uracils, resulting from cytosine deamination, has been detected ubiquitously in aDNA retrieved from diverse sources, and used as an authentication criterion. Here, we employ a library preparation method that separates molecules that carry uracils from those that do not for a set of samples that includes Neandertal remains, herbarium specimens and archaeological plant remains. RESULTS: We show that sequencing DNA libraries enriched in molecules carrying uracils effectively amplifies age associated degradation patterns in microbial mixtures of ancient and historical origin. This facilitates the discovery of authentic ancient microbial taxa in cases where degradation patterns are difficult to detect due to large sequence divergence in microbial mixtures. Additionally, the relative enrichment of taxa in the uracil enriched fraction can help to identify bona fide ancient microbial taxa that could be missed using a more targeted approach. CONCLUSIONS: Our experiments show, that in addition to its use in enriching authentic endogenous DNA of organisms of interest, the selective enrichment of damaged DNA molecules can be a valuable tool in the discovery of ancient microbial taxa.

Ancient Genomes Reveal the Evolutionary History and Origin of Cashmere-Producing Goats in China.

Goats are one of the most widespread farmed animals across the world; however, their migration route to East Asia and local evolutionary history remain poorly understood. Here, we sequenced 27 ancient Chinese goat genomes dating from the Late Neolithic period to the Iron Age. We found close genetic affinities between ancient and modern Chinese goats, demonstrating their genetic continuity. We found that Chinese goats originated from the eastern regions around the Fertile Crescent, and we estimated that the ancestors of Chinese goats diverged from this population in the Chalcolithic period. Modern Chinese goats were divided into a northern and a southern group, coinciding with the most prominent climatic division in China, and two genes related to hair follicle development, FGF5 and EDA2R, were highly divergent between these populations. We identified a likely causal de novo deletion near FGF5 in northern Chinese goats that increased to high frequency over time, whereas EDA2R harbored standing variation dating to the Neolithic. Our findings add to our understanding of the genetic composition and local evolutionary process of Chinese goats.

An optimized method for the extraction of ancient eukaryote DNA from marine sediments.

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo-communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead-beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica-solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size-selection of low molecular-weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead-beating, DNA binding in silica-solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post-library LMW size-selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data-processing protocol should improve quantitative paleo-monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.

Genetic contributions to variation in human stature in prehistoric Europe.

The relative contributions of genetics and environment to temporal and geographic variation in human height remain largely unknown. Ancient DNA has identified changes in genetic ancestry over time, but it is not clear whether those changes in ancestry are associated with changes in height. Here, we directly test whether changes over the past 38,000 y in European height predicted using DNA from 1,071 ancient individuals are consistent with changes observed in 1,159 skeletal remains from comparable populations. We show that the observed decrease in height between the Early Upper Paleolithic and the Mesolithic is qualitatively predicted by genetics. Similarly, both skeletal and genetic height remained constant between the Mesolithic and Neolithic and increased between the Neolithic and Bronze Age. Sitting height changes much less than standing height-consistent with genetic predictions-although genetics predicts a small post-Neolithic increase that is not observed in skeletal remains. Geographic variation in stature is also qualitatively consistent with genetic predictions, particularly with respect to latitude. Finally, we hypothesize that an observed decrease in genetic heel bone mineral density in the Neolithic reflects adaptation to the decreased mobility indicated by decreased femoral bending strength. This study provides a model for interpreting phenotypic changes predicted from ancient DNA and demonstrates how they can be combined with phenotypic measurements to understand the relative contribution of genetic and developmentally plastic responses to environmental change.

An Ancient Harappan Genome Lacks Ancestry from Steppe Pastoralists or Iranian Farmers.

We report an ancient genome from the Indus Valley Civilization (IVC). The individual we sequenced fits as a mixture of people related to ancient Iranians (the largest component) and Southeast Asian hunter-gatherers, a unique profile that matches ancient DNA from 11 genetic outliers from sites in Iran and Turkmenistan in cultural communication with the IVC. These individuals had little if any Steppe pastoralist-derived ancestry, showing that it was not ubiquitous in northwest South Asia during the IVC as it is today. The Iranian-related ancestry in the IVC derives from a lineage leading to early Iranian farmers, herders, and hunter-gatherers before their ancestors separated, contradicting the hypothesis that the shared ancestry between early Iranians and South Asians reflects a large-scale spread of western Iranian farmers east. Instead, sampled ancient genomes from the Iranian plateau and IVC descend from different groups of hunter-gatherers who began farming without being connected by substantial movement of people.

Cytochrome C Oxidase Subunit 1, Internal Transcribed Spacer 1, Nicotinamide Adenine Dinucleotide Hydrogen Dehydrogenase Subunits 2 and 5 of Clonorchis sinensis Ancient DNA Retrieved from Joseon Dynasty Mummy Specimens.

We analyzed Clonorchis sinensis ancient DNA (aDNA) acquired from the specimens of the Joseon mummies. The target regions were cytochrome C oxidase subunit 1 (CO1), internal transcribed spacer 1 (ITS1), nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase subunits 2 (NAD2) and 5 (NAD5). The sequences of C. sinensis aDNA was completely or almost identical to modern C. sinensis sequences in GenBank. We also found that ITS1, NAD2 and NAD5 could be good markers for molecular diagnosis between C. sinensis and the other trematode parasite species. The current result could improve our knowledge about genetic history of C. sinensis.

Setting Up an Ancient DNA Laboratory.

Entering into the world of ancient DNA research is nontrivial. Because the DNA in most ancient specimens is degraded to some extent, the potential is high for contamination of ancient samples, ancient DNA extracts, and genomic sequencing libraries prepared from these extracts with non-degraded DNA from the present-day environment. To minimize the risk of contamination in ancient DNA environments, experimental protocols specific to handling ancient specimens, including those that outline the design and layout of laboratory space, have been introduced. Here, we outline challenges associated with working with ancient samples, including providing guidelines for setting up a new ancient DNA laboratory. We also discuss steps that can be taken at the sample collection and preparation stage to minimize the potential for contamination of ancient DNA experiments with exogenous sources of DNA.

Double-Stranded Library Preparation for Ancient and Other Degraded Samples.

High-throughput sequencing (HTS) allows fast and cost-efficient sequencing of ancient DNA (aDNA) without prior information about what sequences should be targeted. One necessary step for HTS is the preparation of a sequencing library. Commercial kits are available for this purpose, but many of these are not suitable for aDNA or other types of damaged DNA. Here, we outline a protocol for HTS library preparation that is optimized for ancient DNA. We report the library conversion rate for a range of input template and adapter concentrations. Our results show that the protocol performs at a high efficiency.

A Method for Single-Stranded Ancient DNA Library Preparation.

Genomic library preparation from highly degraded DNA is more efficient when library molecules are prepared separately from the complementary strands of DNA fragments. We describe a protocol in which libraries are constructed from single DNA strands in a three-step procedure: single-stranded ligation of the first adapter with T4 DNA ligase in the presence of a splinter oligonucleotide, copying of the DNA strand with a proofreading polymerase, and blunt-end ligation of the second double-stranded adapter with T4 DNA ligase.

Sequencing Library Preparation from Degraded Samples for Non-illumina Sequencing Platforms.

Efficient methods for building genomic sequencing libraries from degraded DNA have been in place for Illumina sequencing platforms for some years now, but such methods are still lacking for other sequencing platforms. Here, we provide a protocol for building genomic libraries from degraded DNA (archival or ancient sample material) for sequencing on the Ion Torrent™ high-throughput sequencing platforms. In addition to a reduction in time and cost in comparison to commercial kits, this protocol removes purification steps prior to library amplification, an important consideration for work involving historical samples. Libraries prepared using this method are appropriate for either shotgun sequencing or enrichment-based downstream approaches.