Allelic bias when performing in-solution enrichment of ancient human DNA

In-solution hybridisation enrichment of genetic variation is a valuable methodology in human paleogenomics. It allows enrichment of endogenous DNA by targeting genetic markers that are comparable between sequencing libraries. Many studies have used the 1240k reagent—which enriches 1,237,207 genome-wide SNPs—since 2015, though access was restricted. In 2021, Twist Biosciences and Daicel Arbor Biosciences independently released commercial kits that enabled all researchers to perform enrichments for the same 1240 k SNPs. We used the Daicel Arbor Biosciences Prime Plus kit to enrich 132 ancient samples from three continents. We identified a systematic assay bias that increases genetic similarity between enriched samples and that cannot be explained by batch effects. We present the impact of the bias on population genetics inferences (e.g. Principal Components Analysis, ƒ-statistics) and genetic relatedness (READ). We compare the Prime Plus bias to that previously reported of the legacy 1240k enrichment assay. In ƒ-statistics, we find that all Prime-Plus-generated data exhibit artefactual excess shared drift, such that within-continent relationships cannot be correctly determined. The bias is more subtle in READ, though interpretation of the results can still be misleading in specific contexts. We expect the bias may affect analyses we have not yet tested. Our observations support previously reported concerns for the integration of different data types in paleogenomics. We also caution that technological solutions to generate 1240k data necessitate a thorough validation process before their adoption in the paleogenomic community.     

Extensive human DNA contamination in extracts from ancient dog bones and teeth

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.     

The use of protein characteristics to assess the retrievability of ancient DNA from ancient bones

The ability to retrieve DNA from ancient specimens has been one of the greatest achievements of the past decade, and has opened a totally new field of research with applications in seemingly distant domains such as archeobotany, the molecular phylogeny of extinct genomes, human paleopathology and the genetic of ancient human populations. However, extraction of ancient DNA has often a very low rate of success, prompting researchers to develop screening methods for the selection of promising specimens. With this goal in mind, we studied the amino acid content of nine human bones of ancient origin. We demonstrate that a single HPLC chromatogram is indicative of the integrity of ancient bone proteins. Among five specimens containing amplifiable DNA, four exhibited a protein content similar to that of contemporary bone protein content. Three of the four specimens, from which we were unable to extract any amplifiable DNA, had an amino acid content strikingly different from that of contem-porary bone. A non-parametric statistical test, Kendall's tau, was used to show that protein content and PCR products, are probably correlated (at a 95% confidence level). In addition, the D/L Asp and D/L Glu racemization ratios obtained are indicative of the presence of ancient organic compounds. We propose that protein analysis should be systematically performed in studies where there are many samples in order to select the specimens that are most likely to contain retrievable ancient DNA.     

Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA

Museum curators and living communities are sometimes reluctant to permit ancient DNA (aDNA) studies of human skeletal remains because the extraction of aDNA usually requires the destruction of at least some skeletal material. Whether these views stem from a desire to conserve precious materials or an objection to destroying ancestral remains, they limit the potential of aDNA research. To help address concerns about destructive analysis and to minimize damage to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains. This method can be used with both teeth and bone, but it preserves the structural integrity of teeth much more effectively than that of bone. Using this method, we demonstrate that it is possible to extract both mitochondrial and nuclear DNA from human remains dating between 300 BC and 1600 AD. Importantly, the method does not expose the remains to hazardous chemicals, allowing them to be safely returned to curators, custodians, and/or owners of the samples. We successfully amplified mitochondrial DNA from 90% of the individuals tested, and we were able to analyze 1-9 nuclear loci in 70% of individuals. We also show that repeated nondestructive extractions from the same tooth can yield amplifiable mitochondrial and nuclear DNA. The high success rate of this method and its ability to yield DNA from samples spanning a wide geographic and temporal range without destroying the structural integrity of the sampled material may make possible the genetic study of skeletal collections that are not available for destructive analysis.     

Sex determination of adolescent skeletons using the distal humerus

Accurate determination of the sex of immature skeletal remains is difficult in the absence of DNA, due to the fact that most sexually dimorphic features of the human skeleton develop as secondary sex characteristics during adolescence. Methods of assessment of adult skeletons cannot reliably be applied to adolescent skeletons because of the transitional nature of the skeleton at puberty and the variability of the adolescent growth spurt. The purpose of this work was to evaluate the accuracy of Rogers's method of morphological sex determination using the distal humerus (Rogers: J Forensic Sci 44 ( 1999 ) 55–59) to assess the sex of adolescent skeletons. The sample consists of 7 documented adolescent skeletons from the Christ Church Spitalfields collection at the British Museum of Natural History and 35 from the Luis Lopes skeletal collection housed in the National History Museum (Museu Bocage) of the University of Lisbon, Portugal. Ages range from 11 to 20 years. The technique achieved an accuracy of 81% on the combined sample of 42. This method can be applied to adolescent skeletons once the trochlea begins fusing to the humeral diaphysis, which occurred by age 11 years in the test samples. Am J Phys Anthropol 2009. © 2009 Wiley‐Liss, Inc.     

Broadening the taxonomic scope of coral reef palaeoecological studies using ancient DNA

Marine environments face acute pressures from human impacts, often resulting in substantial changes in community structure. On the inshore Great Barrier Reef (GBR), palaeoecological studies show the collapse of the previously dominant coral Acropora from the impacts of degraded water quality associated with European colonization. Even more dramatic impacts can result in the replacement of corals by fleshy macroalgae on modern reefs, but their past distribution is unknown because they leave no fossil record. Here, we apply DNA metabarcoding and high‐throughput sequencing of the 18S rDNA gene on palaeoenvironmental DNA (aeDNA) derived from sediment cores at two sites on Pandora Reef (GBR), to enhance palaeoecological studies by incorporating key soft‐bodied taxa, including macroalgae. We compared temporal trends in this aeDNA record with those of coral genera derived from macrofossils. Multivariate analysis of 12 eukaryotic groups from the aeDNA community showed wide variability over the past 750 years. The occurrence of brown macroalgae was negatively correlated only with the dominant coral at both sites. The occurrence of coralline and green macroalgae was positively correlated with only the dominant coral at one of the sites, where we also observed a significant association between the whole coral community and the occurrence of each of the three macroalgae groups. Our results demonstrate that reef sediments can provide a valuable archive for understanding the past distribution and occurrence of important soft‐bodied reef dwellers. Combining information from fossils and aeDNA provides an enhanced understanding of temporal changes of reefs ecosystems at decadal to millennial timescales.     

Improved DNA extraction from ancient bones using silica-based spin columns

We describe a simple method for extracting polymerase chain reaction-amplifiable DNA from ancient bones without the use of organic solvents. Bone powders are digested with proteinase K, and the DNA is purified directly using silica-based spin columns (QIAquick™, QIAGEN). The efficiency of this protocol is demonstrated using human bone samples ranging in age from 15 to 5,000 years old. Am J Phys Anthropol 105:539–543, 1998. © 1998 Wiley-Liss, Inc.     

More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

Authentication of ancient human DNA results is an exceedingly difficult challenge due to the presence of modern contaminant DNA sequences. Nevertheless, the field of ancient human genetics generates huge scientific and public interest, and thus researchers are rarely discouraged by problems concerning the authenticity of such data. Although several methods have been developed to the purpose of authenticating ancient DNA (aDNA) results, while they are useful in faunal research, most of the methods have proven complicated to apply to ancient human DNA. Here, we investigate in detail the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing so, we also produce data that describes the efficiency of bleach incubation of bone powder and its relative detrimental effects on contaminant and authentic ancient DNA. We note that bleach treatment is significantly more detrimental to contaminant than to authentic aDNA in the bleached bone powder. Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination. This asymmetrical behavior of authentic and contaminant DNA can be used to identify authentic haplotypes in human aDNA studies.     

Phylogenetic analysis of ancient mitochondrial DNA lineages of human remains found in Yakutia

Molecular genetic analysis of ancient human remains is mostly based on mtDNA owing to its better preservation in human bones in comparison with nuclear DNA. A study was made of mtDNA extracted from human skeletons found in graves in Yakutia, in order to determine the haplotypes and to compare them with lineages of modern populations. Ancient DNA was extracted from fragments of three skeletons of Yakut graves at At-Dabaan, Ojuluun, and Jaraama sites (dating back to the 18th century) and two skeletons of the Late Neolithic Kerdugen grave (2000–1000 B.C.). All graves were found in central Yakutia (Churapchinskii, Khangalasskii, and Megino-Khangalasskii districts of Yakutia). Five different haplotypes belonging to specific Asian haplogroups were identified. The mtDNA lineages of Yakut graves belong to haplogroups C4a, D5a2, and B5b. The results indicate the continuity of mitochondrial lineages in the Yakut gene pool in the past 300 years. The haplotypes of two humans from the Kerdugen site graves belong to haplogroups A4 and G2a/D. These haplotypes were compared with those of 40000 Eurasian individuals, including 900 from Yakutia. No exact matches were found in Paleo-Asian populations of Chukchi, Eskimos, Koryaks, and Itelmen. Phylogenetically close haplotypes (±1 mutation) were found in Yakut and Evenk populations, as well as in some populations of China and South and West Siberia.     

Mitochondrial DNA analysis of ancient Peruvian highlanders

Ancient DNA recovered from 57 individuals excavated by Hiram Bingham at the rural communities of Paucarcancha, Patallacta, and Huata near the famed Inca royal estate and ritual site of Machu Picchu was analyzed by polymerase chain reaction, and the results were compared with ancient and modern DNA from various Central Andean areas to test their hypothesized indigenous highland origins. The control and coding regions of the mitochondrial DNA (mtDNA) of 35 individuals in this group were sequenced, and the haplogroups of each individual were determined. The frequency data for the haplogroups of these samples show clear proximity to those of modern Quechua and Aymara populations in the Peruvian and Bolivian highlands, and contrast with those of pre-Hispanic individuals of the north coast of Peru that we defined previously. Our study suggests a strong genetic affinity between sampled late pre-Hispanic individuals and modern Andean highlanders. A previous analysis of the Machu Picchu osteological collection suggests that the residents there were a mixed group of natives from various coastal and highland regions relocated by the Inca state for varied purposes. Overall, our study indicates that the sampled individuals from Paucarcancha and Patallacta were indigenous highlanders who provided supportive roles for nearby Machu Picchu.     

The Neandertal genome and ancient DNA authenticity

Recent advances in high‐thoughput DNA sequencing have made genome‐scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large‐scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot‐strap’ approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired.     

Fast and reliable sexing of prosimian and human DNA

Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments, or coamplification of homologous fragments from both sex chromosomes. Existing primers are often unreliable for distantly related species due to mutations in primer regions. Currently there are no published primers for the sexing of prosimian DNA. We show that an existing method (using the zinc finger protein) based on a size difference between the X and Y homologs does not work in prosimians. Multiple alignments of distantly related mammalian species from Genbank and genome databases enabled us to identify conserved regions in the amelogenin gene. Using these conserved regions, we can target species that have no sequence information. We designed a single, conserved primer pair that is useful for fast and reliable molecular sexing of prosimian primates. A single PCR yields two fragments in males and only one in females, which are easily separated with the use of agarose gels. Amplification of separable fragments was successful in seven species of lemurs, as well as humans.     

Amplification of oak DNA from ancient and modern wood

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.     

Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.     

Population origins in Mongolia: genetic structure analysis of ancient and modern DNA

In the present study, nuclear (autosomal and Y-chromosome short tandem repeats) and mitochondrial (hypervariable region I) ancient DNA data previously obtained from a 2,300-year-old Xiongnu population of the Egyin Gol Valley (south of Lake Baikal in northern Mongolia) (Keyser-Tracqui et al. 2003 Am. J. Hum. Genet. 73:247-260) were compared with data from two contemporary Mongolian populations: one from the same location (Egyin Gol Valley plus a perimeter of less than 100 km around the valley), and one from the whole of Mongolia. The principal objective of this comparative analysis was to assess the likelihood that genetic continuity exists between ancient and present-day Mongolian populations. Since the ancient Xiongnu sample might have been composed of some of the ancestors of the present-day Yakuts, data from a present-day Yakut population, as well as published data from Turkish populations, were also included in the comparative analysis. The main result of our study was the genetic similarity observed among Mongolian samples from different periods and geographic areas. This result supports the hypothesis that the succession over time of different Turkic and Mongolian tribes in the current territory of Mongolia resulted in cultural rather than genetic exchanges. Furthermore, it appears that the Yakuts probably did not find their origin among the Xiongnu tribes, as we previously hypothesized.     

Fossil avian eggshell preserves ancient DNA

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.     

A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA

We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP). This is the first ancient nuclear SNP typed from a Native population in the Americas. In addition, using the same teeth we conducted a stable isotopes analysis of collagen and dentine to show that both individuals relied on marine sources (fresh and salt water fish, seals) with no hierarchy seen between them, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots are not hypervariable because they are more prone to damage.