Search Results (25)

Background/Objectives: DNA analysis can be used to expand our understanding of extinct populations and the history of the world and humankind. Dental cavities often contain uncontaminated remains of ancient DNA (aDNA). Archaeological excavations are a convenient source for various samples; however, in almost all extraction methods, a piece of bone or tooth is powdered before extraction, thereby causing damage to archaeological samples that are often irreplaceable and unique. This study aimed to develop a method that enables the collection of DNA from teeth without causing significant damage. Methods: This study presents two methods of DNA extraction from teeth: destructive and non-destructive. Both contemporary and archaeological teeth were examined using both destructive and non-destructive approaches to compare their efficiency. To verify the results, methods such as quantitative RT-PCR, STR analysis, and Y-SNP analysis were employed. Results: Extraction efficiency plays a critical role in this field of research. The main steps of the DNA extraction method were compared and optimized based on purification and using quantitative PCR. Conclusions: The results demonstrate that a non-destructive method of DNA isolation from human teeth can be used successfully, especially when teeth are unique and cannot be destroyed during the examination process. This method yields an appropriate amount of DNA for sequencing. Full article

Human mitochondrial DNA (mtDNA) is a relevant marker in evolutionary and population genetics, including ancient DNA (aDNA) research, due to inherent characteristics. However, aDNA is prone to damage and sequencing artefacts, potentially confounding evolutionary interpretations. To assess evolutionary patterns in ancient and modern mtDNA, we built a phylogeny comprising 63,965 modern and 3757 ancient public mitogenomes, classified mutations by genomic region and functional effect, and analysed distribution, frequency, and predicted pathogenicity of private and pre-terminal mutations, investigating purifying selection. We compared mutation class ratios (non-synonymous, rRNA, tRNA, nonsense vs. synonymous) across ancient and modern terminal branches and pre-terminal nodes. The predicted pathogenicity of non-synonymous mutations was evaluated across major European haplogroups using three tools. Ancient variants exhibited higher ratios of potentially deleterious mutations and significantly elevated pathogenicity scores compared to modern and pre-terminal branches, highlighting a mutation load likely inflated by damage-related artefacts. Remarkably, nonsense mutations—largely incompatible with life—were over 70 times more frequent in aDNA. The correlation between mutation ratios and predicted deleteriousness across haplogroups suggests a pattern incompatible with biological persistence or relaxed selection alone. These findings highlight the importance of rigorous quality control for ancient data in evolutionary inference, molecular clock calibration, and pathogenic variant identification. Full article

Background/Objectives: Ancient DNA (aDNA) research workflows heavily depend on efficient aDNA extraction and NGS library preparation. In this study, we compared some of the commonly used laboratory protocols and compared the source of the bone material for sufficient and reliable results. Methods: We executed a three-phase study. First, we analyzed about 2000 previously processed archaic bone samples and conducted a comparative analysis. The second phase involved a controlled experiment of five ancient individuals, with internal control, to further investigate the efficiency of some of the methods. In the third phase, we made a comparison between the efficiency of two enzymes used for library preparation. Results: Samples made from Pars petrosa resulted in the highest yield of endogenous DNA and longer fragment sizes compared to tooth or skeletal samples. DNA extraction made by MinElute columns preserved slightly longer fragments than the handmade silica suspension. NGS libraries indexed using AccuPrime Pfx produced slightly more consistent insert sizes compared to GoTaq G2. Samples prepared with GoTaq G2 contained slightly more unique molecules. The duplication rates showed no significant impact from enzyme choice. Conclusions: Pars petrosa remains the most reliable source of aDNA, with the extraction method using MinElute columns. While AccuPrime Pfx ensures precise NGS library preparation, a more economical choice of the GoTaq G2 enzyme is a viable alternative for degraded archaic samples. Full article

Ancient human viruses have been detected in ancient DNA (aDNA) samples of both Anatomically Modern Humans and Neanderthals. Reconstructing genomes from aDNA using reference mapping presents numerous problems due to the unique nature of ancient samples, their degraded state, smaller read sizes and the limitations of current methodologies. The spurious alignments of reads to reference sequences (mapping) are a main source of false positives in aDNA assemblies and the assessment of signal-to-noise ratios is essential to differentiate bona fide reconstructions from random, noisy assemblies. Here, we analyzed the statistical distributions of viral genome assemblies, ancient and modern, and their respective random “mock” controls used to evaluate the signal-to-noise ratio. We tested if differences between real and random assemblies could be detected from their statistical distributions. Our analysis shows that the coverage distributions of (1) real viral aDNA assemblies of adenovirus (ADV), herpesvirus (HSV) and papillomavirus (HPV) do not follow power laws nor log-normal laws, (2) (ADV) and control aDNA assemblies are well approximated by log-normal laws, (3) negative control parvovirus B19 (real and random) follow a power law with infinite variance and (4) the mapDamage negative control with non-ancient DNA (modern ADV) and the mapDamage positive control (human mtDNA) are well approximated by the negative binomial distribution, consistent with the Lander–Waterman model. Our results show that the tails of the distributions of aDNA and their controls reveal the weight of random effects and can differentiate spurious assemblies, or false positives, from bona fide assemblies. Full article

The human pathogen Yersinia pestis is responsible for bubonic, septicemic, and pneumonic plague. A deeply comprehensive overview of its historical context, bacteriological characteristics, genomic analysis based on ancient DNA (aDNA) and modern strains, and its impact on historical and actual human populations, is explored. The results from multiple studies have been synthesized to investigate the origins of plague, its transmission, and effects on different populations. Additionally, molecular interactions of Y. pestis, from its evolutionary origins to its adaptation to flea-born transmission, and its impact on human and wild populations are considered. The characteristic combinations of aDNA patterns, which plays a decisive role in the reconstruction and analysis of ancient genomes, are reviewed. Bioinformatics is fundamental in identifying specific Y. pestis lineages, and automated pipelines are among the valuable tools in implementing such studies. Plague, which remains among human history’s most lethal infectious diseases, but also other zoonotic diseases, requires the continuous investigation of plague topics. This can be achieved by improving molecular and genetic screening of animal populations, identifying ecological and social determinants of outbreaks, increasing interdisciplinary collaborations among scientists and public healthcare providers, and continued research into the characterization, diagnosis, and treatment of these diseases. Full article

Paleogenomics focuses on the recovery, manipulation, and analysis of ancient DNA (aDNA) from historical or long-dead organisms to reconstruct and analyze their genomes. The aDNA is commonly obtained from remains found in paleontological and archaeological sites, conserved in museums, and in other archival collections. Herbarium collections represent a great source of phenotypic and genotypic information, and their exploitation has allowed for inference and clarification of previously unsolved taxonomic and systematic relationships. Moreover, herbarium specimens offered a new source for studying phenological traits in plants and for disentangling biogeography and evolutionary scenarios of species. More recently, advances in molecular technologies went in parallel with the decreasing costs of next-generation sequencing (NGS) approaches, which paved the way to the utilization of aDNA for whole-genome studies. Although many studies have been carried out combining modern analytic techniques and ancient samples, such as herbarium specimens, this research field is still relatively unexplored due to the need for improving strategies for aDNA manipulation and exploitation from ancient samples. The higher susceptibility of aDNA to degradation and contamination during herbarium conservation and manipulation and the occurrence of biochemical postmortem damage can result in a more challenging reconstruction of the original DNA sequence. Here, we review the methodological approaches that have been developed for the exploitation of historical herbarium plant materials, such as best practices for aDNA extraction, amplification, and genotyping. We also focus on some strategies to overcome the main problems related to the utilization of herbarium specimens for their exploitation in plant evolutionary studies. Full article

Taxonomic decisions made by palaeontologists are often based on a few morphological features preserved in the fossil material. This practice may sometimes lead to the description of new species based on single specimens, which are, in fact, extreme or aberrant morphological variants of known taxa. Ancient DNA (aDNA) analysis of the Late Pleistocene specimens from the archaeological site Petits Guinards (Creuzier-la-Vieux, Allier, France), described as a new vole Microtus (Sumeriomys) bifrons, did not confirm the species distinctness of the studied population. The genetically examined specimens belonged to Stenocranius anglicus and/or Microtus arvalis, the dominant species at the site. Our findings show that it is risky to describe new fossil taxa on the basis of phenotypic outliers or morphologically aberrant, rare specimens that do not fall within the previously known population variability. We also highlight the importance of ancient DNA in resolving taxonomic and nomenclature problems and classifying fossil mammals of the Late Pleistocene age. Full article

Ancient DNA (aDNA) research first began in 1984 and ever since has greatly expanded our understanding of evolution and migration. Today, aDNA analysis is used to solve various puzzles about the origin of mankind, migration patterns, and the spread of infectious diseases. The incredible findings ranging from identifying the new branches within the human family to studying the genomes of extinct flora and fauna have caught the world by surprise in recent times. However, a closer look at these published results points out a clear Global North and Global South divide. Therefore, through this research, we aim to emphasize encouraging better collaborative opportunities and technology transfer to support researchers in the Global South. Further, the present research also focuses on expanding the scope of the ongoing conversation in the field of aDNA by reporting relevant literature published around the world and discussing the advancements and challenges in the field. Full article

Paleogenetics has significantly changed since its inception almost forty years ago. Initially, molecular techniques available to the researchers offered minimal possibilities for ancient DNA analysis. The subsequent expansion of the scientific tool cabinet allowed for more remarkable achievements, combined has with the newfound popularity of this budding field of science. Finally, a breakthrough was made with the development of next-generation sequencing (NGS) technologies and the update of DNA isolation protocols, through which even very fragmented aDNA samples could be used to sequence whole genomes. In this paper, we review the achievements made thus far and compare the methodologies utilized in this field of science, discussing their benefits and challenges. Full article

Gray wolves (Canis lupus) in the Iberian Peninsula declined substantially in both range and population size in the last few centuries due to human persecution and habitat fragmentation. However, unlike many other western European populations, gray wolves never went extinct in Iberia. Since the minimum number was recorded around 1970, their numbers have significantly increased and then stabilized in recent decades. We analyzed mitochondrial genomes from 54 historical specimens of Iberian wolves from across their historical range using ancient DNA methods. We compared historical and current mitochondrial diversity in Iberian wolves at the 5′ end of the control region (n = 17 and 27) and the whole mitochondrial genome excluding the control region (n = 19 and 29). Despite an increase in population size since the 1970s, genetic diversity declined. We identified 10 whole mitochondrial DNA haplotypes in 19 historical specimens, whereas only six of them were observed in 29 modern Iberian wolves. Moreover, a haplotype that was restricted to the southern part of the distribution has gone extinct. Our results illustrate a lag between demographic and genetic diversity changes, and show that after severe population declines, genetic diversity can continue to be lost in stable or even expanding populations. This suggests that such populations may be of conservation concern even after their demographic trajectory has been reversed. Full article

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. Full article

Ancient DNA from, Neandertal and modern human fossils, and comparative morphological analyses of them, reveal a complex history of interbreeding between these lineages and the introgression of Neandertal genes into modern human genomes. Despite substantial increases in our knowledge of these events, the timing and geographic location of hybridization events remain unclear. Six measures of facial size and shape, from regional samples of Neandertals and early modern humans, were used in a multivariate exploratory analysis to try to identify regions in which early modern human facial morphology was more similar to that of Neandertals, which might thus represent regions of greater introgression of Neandertal genes. The results of canonical variates analysis and hierarchical cluster analysis suggest important affinities in facial morphology between both Middle and Upper Paleolithic early modern humans of the Near East with Neandertals, highlighting the importance of this region for interbreeding between the two lineages. Full article

Retrieving high-quality endogenous ancient DNA (aDNA) poses several challenges, including low molecular copy number, high rates of fragmentation, damage at read termini, and potential presence of exogenous contaminant DNA. All these factors complicate a reliable reconstruction of consensus aDNA sequences in reads from high-throughput sequencing platforms. Here, we report findings from a thorough evaluation of two alternative tools (ANGSD and schmutzi) aimed at overcoming these issues and constructing high-quality ancient mitogenomes. Raw genomic data (BAM/FASTQ) from a total of 17 previously published whole ancient human genomes ranging from the 14th to the 7th millennium BCE were retrieved and mitochondrial consensus sequences were reconstructed using different quality filters, with their accuracy measured and compared. Moreover, the influence of different sequence parameters (number of reads, sequenced bases, mean coverage, and rate of deamination and contamination) as predictors of derived sequence quality was evaluated. Complete mitogenomes were successfully reconstructed for all ancient samples, and for the majority of them, filtering substantially improved mtDNA consensus calling and haplogroup prediction. Overall, the schmutzi pipeline, which estimates and takes into consideration exogenous contamination, appeared to have the edge over the much faster and user-friendly alternative method (ANGSD) in moderate to high coverage samples (>1,000,000 reads). ANGSD, however, through its read termini trimming filter, showed better capabilities in calling the consensus sequence from low-quality samples. Among all the predictors of overall sample quality examined, the strongest correlation was found for the available number of sequence reads and bases. In the process, we report a previously unassigned haplogroup (U3b) for an Early Chalcolithic individual from Southern Anatolia/Northern Levant. Full article

Fungi are ecologically important in several ecosystem processes, yet their community composition, ecophysiological roles, and responses to changing environmental factors in historical sediments are rarely studied. Here we explored ancient fungal DNA from lake Lielais Svētiņu sediment throughout the Holocene (10.5 kyr) using the ITS metabarcoding approach. Our data revealed diverse fungal taxa and smooth community changes during most of the Holocene with rapid changes occurring in the last few millennia. More precisely, plankton parasitic fungi became more diverse from the Late Holocene (2–4 kyr) which could be related to a shift towards a cooler climate. The Latest Holocene (~2 kyr) showed a distinct increase in the richness of plankton parasites, mycorrhizal, and plant pathogenic fungi which can be associated with an increased transfer rate of plant material into the lake and blooms of planktonic organisms influenced by increased, yet moderate, human impact. Thus, major community shifts in plankton parasites and mycorrhizal fungi could be utilized as potential paleo-variables that accompany host-substrate dynamics. Our work demonstrates that fungal aDNA with predicted ecophysiology and host specificity can be employed to reconstruct both aquatic and surrounding terrestrial ecosystems and to estimate the influence of environmental change. Full article

We aimed to extract DNA and amplify PCR fragments at the mitochondrial DNA Nad7.1 locus and 11 nuclear microsatellite loci in nine circa 11,000-year-old individuals of Scots pine found at the bottom of the Baltic sea and test the genetic associations with the present-day gene pool of Scots pine in Lithuania. We followed a strict anticontamination protocol in the lab and, simultaneously with the aDNA specimens, tested DNA-free controls. The DNA was extracted by an ATMAB protocol from the ancient wood specimens sampled underwater from Scots pine stumps located circa 20–30 m deep and circa 12 km ashore in western Lithuania. As the references, we used 30 present-day Lithuanian populations of Scots pine with 25–50 individuals each. The aDNA yield was 11–41 ng/μL. The PCR amplification for the mtDNA Nad7.1 locus and the nDNA loci yielded reliable aDNA fragments for three and seven out of nine ancient pines, respectively. The electrophoresis profiles of all the PCR tested DNA-free controls contained the sizing standard only, indicating low likelihood for contamination. At the mtDNA Nad7.1 locus, all three ancient Scots pine individuals had the type A (300 bp) allele, indicating postglacial migration from the refugia in Balkan peninsula. The GENECLASS Bayesian assignment tests revealed relatively stringer and consistent genetic associations between the ancient Scots pine trees and the present-day southern Lithuanian populations (assignment probability 0.37–0.55) and several wetlands in Lithuania. Our study shows that salty sea water efficiently preserves ancient DNA in wood at the quality levels suitable for genetic testing of trees dated back as far as 11,000 years before present. Full article