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.     

Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

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.     

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.     

Sex determination of ancient human skeletons using DNA

A method for determining the sex of human skeletons was developed using molecular genetic techniques. The amelogenin gene, found on the X and Y chromosomes, was examined using the polymerase chain reaction (PCR) and a nonradioactive dot blot procedure. DNA was analyzed from 20 modern individuals of known sex and 20 skeletons from an archaeological site in central Illinois dating to A.D. 1300. An independent assessment of the sex of each skeleton was made according to standard osteological methods. The sex of 19 ancient and 20 modern individuals was accurately determined using this molecular genetic technique. Molecular sex determination will be especially useful for juvenile and fragmentary remains when it is difficult, or impossible, to establish an individual's sex from morphological features. © 1996 Wiley-Liss, Inc.     

Palaeontology in a molecular world: the search for authentic ancient DNA

The survival of ancient DNA in specimens up to several thousands of years old is established. However, there have been several claims concerning the recovery of geologically ancient DNA from fossil material many millions of years old. The authenticity of these fossil DNA sequences is questionable on theoretical and empirical grounds, and the existence of authentic geologically ancient DNA remains to be proven.     

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.     

Reconstructing the evolutionary history of China: a caveat about inferences drawn from ancient DNA

The decipherment of the meager information provided by short fragments of ancient mitochondrial DNA (mtDNA) is notoriously difficult but is regarded as a most promising way toward reconstructing the past from the genetic perspective. By haplogroup-specific hypervariable segment (HVS) motif search and matching or near-matching with available modern data sets, most of the ancient mtDNAs can be tentatively assigned to haplogroups, which are often subcontinent specific. Further typing for mtDNA haplogroup-diagnostic coding region polymorphisms, however, is indispensable for establishing the geographic/genetic affinities of ancient samples with less ambiguity. In the present study, we sequenced a fragment (approximately 982 bp) of the mtDNA control region in 76 Han individuals from Taian, Shandong, China, and we combined these data with previously reported samples from Zibo and Qingdao, Shandong. The reanalysis of two previously published ancient mtDNA population data sets from Linzi (same province) then indicates that the ancient populations had features in common with the modern populations from south China rather than any specific affinity to the European mtDNA pool. Our results highlight that ancient mtDNA data obtained under different sampling schemes and subject to potential contamination can easily create the impression of drastic spatiotemporal changes in the genetic structure of a regional population during the past few thousand years if inappropriate methods of data analysis are employed.     

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.     

Evidence of ancient DNA reveals the first European lineage in Iron Age Central China

Various studies on ancient DNA have attempted to reconstruct population movement in Asia, with much interest focused on determining the arrival of European lineages in ancient East Asia. Here, we discuss our analysis of the mitochondrial DNA of human remains excavated from the Yu Hong tomb in Taiyuan, China, dated 1400 years ago. The burial style of this tomb is characteristic of Central Asia at that time. Our analysis shows that Yu Hong belonged to the haplogroup U5, one of the oldest western Eurasian-specific haplogroups, while his wife can be classified as haplogroup G, the type prevalent in East Asia. Our findings show that this man with European lineage arrived in Taiyuan approximately 1400 years ago, and most probably married a local woman. Haplogroup U5 was the first west Eurasian-specific lineage to be found in the central part of ancient China, and Taiyuan may be the easternmost location of the discovered remains of European lineage in ancient China.     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Parallel detection of ancient pathogens via array-based DNA capture

DNA capture coupled with next generation sequencing is highly suitable for the study of ancient pathogens. Screening for pathogens can, however, be meticulous when assays are restricted to the enrichment of single organisms, which is common practice. Here, we report on an array-based DNA capture screening technique for the parallel detection of nearly 100 pathogens that could have potentially left behind molecular signatures in preserved ancient tissues. We demonstrate the sensitivity of our method through evaluation of its performance with a library known to harbour ancient Mycobacterium leprae DNA. This rapid and economical technique will be highly useful for the identification of historical diseases that are difficult to characterize based on archaeological information alone.     

DNA content and distribution in ancient feathers and potential to reconstruct the plumage of extinct avian taxa

Feathers are known to contain amplifiable DNA at their base (calamus) and have provided an important genetic source from museum specimens. However, feathers in subfossil deposits generally only preserve the upper shaft and feather ‘vane’ which are thought to be unsuitable for DNA analysis. We analyse subfossil moa feathers from Holocene New Zealand rockshelter sites and demonstrate that both ancient DNA and plumage information can be recovered from their upper portion, allowing species identification and a means to reconstruct the appearance of extinct taxa. These ancient DNA sequences indicate that the distal portions of feathers are an untapped resource for studies of museum, palaeontological and modern specimens. We investigate the potential to reconstruct the plumage of pre-historically extinct avian taxa using subfossil remains, rather than assuming morphological uniformity with closely related extant taxa. To test the notion of colour persistence in subfossil feathers, we perform digital comparisons of feathers of the red-crowned parakeet (Cyanoramphus novaezelandiae novaezelandiae) excavated from the same horizons as the moa feathers, with modern samples. The results suggest that the coloration of the moa feathers is authentic, and computer software is used to perform plumage reconstructions of moa based on subfossil remains.     

Endophytic fungal DNA, the source of contamination in spruce needle DNA

DNA isolated and amplified from higher plants may originate from symbiotic microbes occupying plant tissues. A recent report on the phylogeny of Picea contained sequence data that upon later analysis proved to originate from filamentous ascomycetes. Isolates of endophytic fungi from Picea foliage collected from the same location as the original samples were examined to identify the source of the contaminating DNA. The ITS region of isolates was screened by Southern blotting using an oligonucleotide probe homologous to a unique portion of the reported 'spruce' sequences. This study identifies a DNA sequence originally attributed to Picea engelmannii (Engelmann spruce) as Hormonema dematioides , a ubiquitous foliar endophyte of conifers. Infections of plants by endophytic fungi are common and their presence is not revealed by external symptoms. Plant molecular researchers should be aware of the potential for this type of DNA contamination.     

Proxy comparison in ancient peat sediments: pollen,macrofossil and plant DNA

We compared DNA, pollen and macrofossil data obtained from Weichselian interstadial (age more than 40 kyr) and Holocene (maximum age 8400 cal yr BP) peat sediments from northern Europe and used them to reconstruct contemporary floristic compositions at two sites. The majority of the samples provided plant DNA sequences of good quality with success amplification rates depending on age. DNA and sequencing analysis provided five plant taxa from the older site and nine taxa from the younger site, corresponding to 7% and 15% of the total number of taxa identified by the three proxies together. At both sites, pollen analysis detected the largest (54) and DNA the lowest (10) number of taxa, but five of the DNA taxa were not detected by pollen and macrofossils. The finding of a larger overlap between DNA and pollen than between DNA and macrofossils proxies seems to go against our previous suggestion based on lacustrine sediments that DNA originates principally from plant tissues and less from pollen. At both sites, we also detected Quercus spp. DNA, but few pollen grains were found in the record, and these are normally interpreted as long-distance dispersal. We confirm that in palaeoecological investigations, sedimentary DNA analysis is less comprehensive than classical morphological analysis, but is a complementary and important tool to obtain a more complete picture of past flora.     

Angiosperm DNA contamination by endophytic fungi: Detection and methods of avoidance

PCR primers with broad applicability are useful in many molecular-based studies; however, their universality can compromise results when DNA contaminants also are amplified. Eighty-one templates ofDahlia (Asteraceae), primarily extracted from native Mexican populations, were tested for the presence of fungal contaminants; out of these, almost 1 in 7 templates (13.6%) was contaminated. In a second survey across 12 angiosperm families using material collected in Illinois, fungal DNA contaminated over 60% of the templates analyzed. Endophytic fungi often are symptomless symbionts living within the above-ground tissues of their angiosperm hosts and are not affected by surface sterilization techniques. Recent studies have revealed their widespread occurrence and broad host range. We also present field strategies for obtaining plant material to reduce the possibility of collecting infected leaves and a simple screening test for detecting fungal DNA in angiosperm templates.     

Elevated substitution rate estimates from ancient DNA: model violation and bias of Bayesian methods

The increasing ability to extract and sequence DNA from noncontemporaneous tissue offers biologists the opportunity to analyse ancient DNA (aDNA) together with modern DNA (mDNA) to address the taxonomy of extinct species, evolutionary origins, historical phylogeography and biogeography. Perhaps more exciting are recent developments in coalescence-based Bayesian inference that offer the potential to use temporal information from aDNA and mDNA for the estimation of substitution rates and divergence dates as an alternative to fossil and geological calibration. This comes at a time of growing interest in the possibility of time dependency for molecular rate estimates. In this study, we provide a critical assessment of Bayesian Markov chain Monte Carlo (MCMC) analysis for the estimation of substitution rate using simulated samples of aDNA and mDNA. We conclude that the current models and priors employed in Bayesian MCMC analysis of heterochronous mtDNA are susceptible to an upward bias in the estimation of substitution rates because of model misspecification when the data come from populations with less than simple demographic histories, including sudden short-lived population bottlenecks or pronounced population structure. However, when model misspecification is only mild, then the 95% highest posterior density intervals provide adequate frequentist coverage of the true rates.     

The use of herbarium specimens in DNA phylogenetics: Evaluation and improvement

During the last few years we have been confronted with the need to use herbarium specimens in the molecular phylogeny studies, since it is generally difficult to obtain living material of some rare species. Ancient DNA has been sequenced, and there are also reports on successful DNA amplification from herbarium specimens. However, it is not easy to obtain amplified DNA from the first herbarium sample tested. In this paper, experiments are described about trials of DNA amplification from two to 151-year-old herbarium specimens of plant species we needed for our projects. Of the 17 herbarium samples tested only two allowed DNA amplification under standard DNA isolation conditions. Different types of PCR inhibiting activities were demonstrated in DNA extracts. In some of the extracts there was extremely low concentration of template with satisfactory quality. In some instances, PCR inhibiting activities were successfully removed by treating them either with insoluble polyvinylpyrrolidone or by adding bovine serum albumin (BSA) to the amplification mixture. However, some PCR-inhibiting activities were resistant to the treatments described above. When the concentration of template was very low, a second PCR amplification with internal primers was necessary to increase the amount of DNA for sequencing. Nevertheless, contamination of either DNA extract or amplification mixture were sometimes observed, and consequently precautions were taken to minimize them. Finally, successful amplification was obtained in eight samples out of the 17 examined.