Improving ancient DNA read mapping against modern reference genomes

ABSTRACT: BACKGROUND: Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. RESULTS: In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. CONCLUSIONS: We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.     

Ancient genomics

The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field''s focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past.     

The De Novo Assembly of Mitochondrial Genomes of the Extinct Passenger Pigeon (Ectopistes migratorius) with Next Generation Sequencing

The information from ancient DNA (aDNA) provides an unparalleled opportunity to infer phylogenetic relationships and population history of extinct species and to investigate genetic evolution directly. However, the degraded and fragmented nature of aDNA has posed technical challenges for studies based on conventional PCR amplification. In this study, we present an approach based on next generation sequencing to efficiently sequence the complete mitochondrial genome (mitogenome) of two extinct passenger pigeons (Ectopistes migratorius) using de novo assembly of massive short (90 bp), paired-end or single-end reads. Although varying levels of human contamination and low levels of postmortem nucleotide lesion were observed, they did not impact sequencing accuracy. Our results demonstrated that the de novo assembly of shotgun sequence reads could be a potent approach to sequence mitogenomes, and offered an efficient way to infer evolutionary history of extinct species.     

Resurrecting ancient animal genomes: the extinct moa and more

Recently two developments have had a major impact on the field of ancient DNA (aDNA). First, new advances in DNA sequencing, in combination with improved capture/enrichment methods, have resulted in the recovery of orders of magnitude more DNA sequence data from ancient animals. Second, there has been an increase in the range of tissue types employed in aDNA. Hair in particular has proven to be very successful as a source of DNA because of its low levels of contamination and high level of ancient endogenous DNA. These developments have resulted in significant advances in our understanding of recently extinct animals: namely their evolutionary relationships, physiology, and even behaviour. Hair has been used to recover the first complete ancient nuclear genome, that of the extinct woolly mammoth, which then facilitated the expression and functional analysis of haemoglobins. Finally, we speculate on the consequences of these developments for the possibility of recreating extinct animals.     

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.     

Damage and repair of ancient DNA

Under certain conditions small amounts of DNA can survive for long periods of time and can be used as polymerase chain reaction (PCR) substrates for the study of phylogenetic relationships and population genetics of extinct plants and animals, including hominids. Because of extensive DNA degradation, these studies are limited to species that lived within the past 10(4)-10(5) years (Late Pleistocene), although DNA sequences from 10(6) years have been reported. Ancient DNA (aDNA) has been used to study phylogenetic relationships of protists, fungi, algae, plants, and higher eukaryotes such as extinct horses, cave bears, the marsupial wolf, the moa, and Neanderthal. In the past few years, this technology has been extended to the study of infectious disease in ancient Egyptian and South American mummies, the dietary habits of ancient animals, and agricultural practices and population dynamics of early native Americans. Hence, ancient DNA contains information pertinent to numerous fields of study including evolution, population genetics, ecology, climatology, medicine, archeology, and behavior. The major obstacles to the study of aDNA are its extremely low yield, contamination with modern DNA, and extensive degradation. In the course of this review, we will discuss the current aDNA literature describing the importance of aDNA studies as they relate to important biological questions and the difficulties associated with extracting useful information from highly degraded and damaged substrates derived from limited sources. In addition, we will present some of our own preliminary and published data on mechanisms of DNA degradation and some speculative thoughts on strategies for repair and restoration of aDNA.     

Ancient DNA analysis of human populations

The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800-1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0-100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA.     

Ancient DNA complements microfossil record in deep-sea subsurface sediments

Deep-sea subsurface sediments are the most important archives of marine biodiversity. Until now, these archives were studied mainly using the microfossil record, disregarding large amounts of DNA accumulated on the deep-sea floor. Accessing ancient DNA (aDNA) molecules preserved down-core would offer unique insights into the history of marine biodiversity, including both fossilized and non-fossilized taxa. Here, we recover aDNA of eukaryotic origin across four cores collected at abyssal depths in the South Atlantic, in up to 32.5 thousand-year-old sediment layers. Our study focuses on Foraminifera and Radiolaria, two major groups of marine microfossils also comprising diverse non-fossilized taxa. We describe their assemblages in down-core sediment layers applying both micropalaeontological and environmental DNA sequencing approaches. Short fragments of the foraminiferal and radiolarian small subunit rRNA gene recovered from sedimentary DNA extracts provide evidence that eukaryotic aDNA is preserved in deep-sea sediments encompassing the last glacial maximum. Most aDNA were assigned to non-fossilized taxa that also dominate in molecular studies of modern environments. Our study reveals the potential of aDNA to better document the evolution of past marine ecosystems and opens new horizons for the development of deep-sea palaeogenomics.     

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.     

Identification of ancient Olea europaea L. and Cornus mas L. seeds by DNA barcoding

The analysis of ancient DNA (aDNA) provides archaeologists and anthropologists with innovative, scientific and accurate data to study and understand the past. In this work, ancient seeds, found in the "Mora Cavorso" archaeological site (Latium, Central Italy), were analyzed to increase information about Italian Neolithic populations (plant use, agriculture, diet, trades, customs and ecology). We performed morphological and genetic techniques to identify fossil botanical species. In particular, this study also suggests and emphasizes the use of DNA barcode method for ancient plant sample analysis. Scanning electron microscope (SEM) observations showed seed compact structure and irregular surface but they did not permit a precise nor empirical classification: so, a molecular approach was necessary. DNA was extracted from ancient seeds and then it was used, as template, for PCR amplifications of standardized barcode genes. Although aDNA could be highly degraded by the time, successful PCR products were obtained, sequenced and compared to nucleotide sequence databases. Positive outcomes (supported by morphological comparison with modern seeds, geographical distribution and historical data) indicated that seeds could be identified as belonging to two plant species: Olea europaea L. and Cornus mas L.     

Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology

Identifying the causes of past epidemics depends on the specific detection of pathogens in buried individuals; this field of research is known as paleomicrobiology, an emerging field that has benefited from technological advances in microbiology. For almost 15 years, the detection, identification, and characterization of microbes in ancient environmental and human specimens emerged on the basis of ancient DNA (aDNA) analyses. aDNA limitations due to potential contamination by modern DNA and altered aDNA led to the development of alternative methods for the detection and characterization of nonnucleotidic biomolecules, including mycolic acids (of ancient mycobacteria) and proteins. Accordingly, immunohistochemistry, immunochromatography, and enzyme-linked immunosorbent assay techniques have been developed for the specific detection of microbes from ancient human and environmental specimens. Protein analysis by mass spectrometry, a standard for ancient animal identification, has also recently emerged as a technique for ancient mycobacteria detection, while immuno-PCR is yet another promising technique. As with aDNA, strict protocols must be enforced to ensure authenticity of the data. Here we review the analysis of nonnucleotidic biomolecules from ancient microbes and the ability of these analyses to complement aDNA analyses, which opens new opportunities for identification of ancient microbes as well as new avenues to potentially resolve controversies regarding the cause of some historical pandemics and study the coevolution of microbes and hosts.     

Ancient DNA sheds new light on the Svalbard foraminiferal fossil record of the last millennium

Recent palaeogenetic studies have demonstrated the occurrence of preserved ancient DNA (aDNA) in various types of fossilised material. Environmental aDNA sequences assigned to modern species have been recovered from marine sediments dating to the Pleistocene. However, the match between the aDNA and the fossil record still needs to be evaluated for the environmental DNA approaches to be fully exploited. Here, we focus on foraminifera in sediments up to one thousand years old retrieved from the Hornsund fjord (Svalbard). We compared the diversity of foraminiferal microfossil assemblages with the diversity of aDNA sequenced from subsurface sediment samples using both cloning and high‐throughput sequencing (HTS). Our study shows that 57% of the species archived in the fossil record were also detected in the aDNA data. However, the relative abundance of aDNA sequence reads and fossil specimens differed considerably. We also found a limited match between the stratigraphic occurrence of some fossil species and their aDNA sequences, especially in the case of rare taxa. The aDNA data comprised a high proportion of non‐fossilised monothalamous species, which are known to dominate in modern foraminiferal communities of the Svalbard region. Our results confirm the relevance of HTS for studying past micro‐eukaryotic diversity and provide insight into its ability to reflect fossil assemblages. Palaeogenetic studies including aDNA analyses of non‐fossilised groups expand the range of palaeoceanographical proxies and therefore may increase the accuracy of palaeoenvironmental reconstructions.     

Yersinia pestis: New Evidence for an Old Infection

The successful reconstruction of an ancient bacterial genome from archaeological material presents an important methodological advancement for infectious disease research. The reliability of evolutionary histories inferred by the incorporation of ancient data, however, are highly contingent upon the level of genetic diversity represented in modern genomic sequences that are publicly accessible, and the paucity of available complete genomes restricts the level of phylogenetic resolution that can be obtained. Here we add to our original analysis of the Yersinia pestis strain implicated in the Black Death by consolidating our dataset for 18 modern genomes with single nucleotide polymorphism (SNP) data for an additional 289 strains at over 600 positions. The inclusion of this additional data reveals a cluster of Y. pestis strains that diverge at a time significantly in advance of the Black Death, with divergence dates roughly coincident with the Plague of Justinian (6^th to 8^th century AD). In addition, the analysis reveals further clues regarding potential radiation events that occurred immediately preceding the Black Death, and the legacy it may have left in modern Y. pestis populations. This work reiterates the need for more publicly available complete genomes, both modern and ancient, to achieve an accurate understanding of the history of this bacterium.     

Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

Despite the abundance of fossil remains for the extinct steppe bison (Bison priscus), an animal that was painted and engraved in numerous European Paleolithic caves, a complete mitochondrial genome sequence has never been obtained for this species. In the present study we collected bone samples from a sector of the Trois-Frères Paleolithic cave (Ariège, France) that formerly functioned as a pitfall and was sealed before the end of the Pleistocene. Screening the DNA content of the samples collected from the ground surface revealed their contamination by Bos DNA. However, a 19,000-year-old rib collected on a rock apart the pathway delineated for modern visitors was devoid of such contaminants and reproducibly yielded Bison priscus DNA. High-throughput shotgun sequencing combined with conventional PCR analysis of the rib DNA extract enabled to reconstruct a complete mitochondrial genome sequence of 16,318 bp for the extinct steppe bison with a 10.4-fold coverage. Phylogenetic analyses robustly established the position of the Bison priscus mitochondrial genome as basal to the clade delineated by the genomes of the modern American Bison bison. The extinct steppe bison sequence, which exhibits 93 specific polymorphisms as compared to the published Bison bison mitochondrial genomes, provides an additional resource for the study of Bovinae specimens. Moreover this study of ancient DNA delineates a new research pathway for the analysis of the Magdalenian Trois-Frères cave.     

An in vitro strategy for the selective isolation of anomalous DNA from prokaryotic genomes

In sequenced genomes of prokaryotes, anomalous DNA (aDNA) can be recognized, among others, by atypical clustering of dinucleotides. We hypothesized that atypical clustering of hexameric endonuclease recognition sites in aDNA allows the specific isolation of anomalous sequences in vitro. Clustering of endonuclease recognition sites in aDNA regions of eight published prokaryotic genome sequences was demonstrated. In silico digestion of the Neisseria meningitidis MC58 genome, using four selected endonucleases, revealed that out of 27 of the small fragments predicted (<5 kb), 21 were located in known genomic islands. Of the 24 calculated fragments (>300 bp and <5 kb), 22 met our criteria for aDNA, i.e. a high dinucleotide dissimilarity and/or aberrant GC content. The four enzymes also allowed the identification of aDNA fragments from the related Z2491 strain. Similarly, the sequenced genomes of three strains of Escherichia coli assessed by in silico digestion using XbaI yielded strain-specific sets of fragments of anomalous composition. In vitro applicability of the method was demonstrated by using adaptor-linked PCR, yielding the predicted fragments from the N.meningitidis MC58 genome. In conclusion, this strategy allows the selective isolation of aDNA from prokaryotic genomes by a simple restriction digest–amplification–cloning–sequencing scheme.     

Evolutionary anthropology and genes: Investigating the genetics of human evolution from excavated skeletal remains

The development of molecular tools for the extraction, analysis and interpretation of DNA from the remains of ancient organisms (paleogenetics) has revolutionised a range of disciplines as diverse as the fields of human evolution, bioarchaeology, epidemiology, microbiology, taxonomy and population genetics. The paper draws attention to some of the challenges associated with the extraction and interpretation of ancient DNA from archaeological material, and then reviews the influence of paleogenetics on the field of human evolution. It discusses the main contributions of molecular studies to reconstructing the evolutionary and phylogenetic relationships between extinct hominins (human ancestors) and anatomically modern humans. It also explores the evidence for evolutionary changes in the genetic structure of anatomically modern humans in recent millennia. This breadth of research has led to discoveries that would never have been possible using traditional approaches to human evolution.     

Application de la technique de PCR en temps réel à l’étude de l’ADN ancien

Real-time Polymerase Chain Reaction (PCR) for the study of ancient DNA. The properties of ancient DNA (aDNA) make difficult the retrieval of DNA sequence. The advantage of Real-Time PCR was exploited, for the first time, in the study of aDNA. We determined the optimal condition to amplify, in one round of PCR, aDNA, which should be directly sequenced. Beside the verification of aDNA authenticity, we compared two cleaning bone methods: scalpel and ethanol. The ethanol specimens showed the best DNA yield. The aDNA was extracted and amplified (mitochondrial hypervariable region I) from five skeletons exhumed from the archaeological site of Notre-Dame-du-Bourg (France), dated from 3rd to 17th century. To cite this article: R. Kefi et al., C. R. Palevol 2 (2003) 125–132.     

Ancestral Polymorphisms and Sex-Biased Migration Shaped the Demographic History of Brown Bears and Polar Bears

Recent studies have reported discordant gene trees in the evolution of brown bears and polar bears. Genealogical histories are different among independent nuclear loci and between biparentally inherited autosomal DNA (aDNA) and matrilineal mitochondrial DNA (mtDNA). Based on multi-locus genomic sequences from aDNA and mtDNA, we inferred the population demography of brown and polar bears and found that brown bears have 6 times (aDNA) or more than 14 times (mtDNA) larger population sizes than polar bears and that polar bear lineage is derived from within brown bear diversity. In brown bears, the effective population size ratio of mtDNA to aDNA was at least 0.62, which deviated from the expected value of 0.25, suggesting matriarchal population due to female philopatry and male-biased migration. These results emphasize that ancestral polymorphisms and sex-biased migration may have contributed to conflicting branching patterns in brown and polar bears across aDNA genes and mtDNA.     

Diversification of Sulawesi macaque monkeys: decoupled evolution of mitochondrial and autosomal DNA

Abstract. In macaque monkeys, females are philopatric and males are obligate dispersers. This social system is expected to differently affect evolution of genetic elements depending on their mode of inheritance. Because of this, the geographic structure of molecular variation may differ considerably in mitochondrial DNA (mtDNA) and in autosomal DNA (aDNA) in the same individuals, even though these genomes are partially co-inherited. On the Indonesian island of Sulawesi, macaque monkeys underwent an explosive diversification as a result of range fragmentation. Today, barriers to dispersal have receded and fertile hybrid individuals can be found at contact zones between parapatric species. In this study, we examine the impact of range fragmentation on Sulawesi macaque mtDNA and aDNA by comparing evolution, phylogeography, and population subdivision of each genome. Our results suggest that mtDNA is paraphyletic in some species, and that mtDNA phylogeography is largely consistent with a pattern of isolation by distance. Autosomal DNA, however, is suggestive of fragmentation, in that interspecific differentiation across most contact zones is significant but intraspecific differentiation between contact zones is not. Furthermore, in mtDNA, most molecular variation is partitioned between populations within species but in aDNA most variation is partitioned within populations. That mtDNA has a different geographic structure than aDNA (and morphology) in these primates is a probable consequence of (1) a high level of ancestral polymorphism in mtDNA, (2) differences between patterns of ancestral dispersal of matrilines and contemporary dispersal of males, and (3) the fact that female philopatry impedes gene flow of macaque mtDNA.     

Genome data from a sixteenth century pig illuminate modern breed relationships

Ancient DNA (aDNA) provides direct evidence of historical events that have modeled the genome of modern individuals. In livestock, resolving the differences between the effects of initial domestication and of subsequent modern breeding is not straight forward without aDNA data. Here, we have obtained shotgun genome sequence data from a sixteenth century pig from Northeastern Spain (Montsoriu castle), the ancient pig was obtained from an extremely well-preserved and diverse assemblage. In addition, we provide the sequence of three new modern genomes from an Iberian pig, Spanish wild boar and a Guatemalan Creole pig. Comparison with both mitochondrial and autosomal genome data shows that the ancient pig is closely related to extant Iberian pigs and to European wild boar. Although the ancient sample was clearly domestic, admixture with wild boar also occurred, according to the D-statistics. The close relationship between Iberian, European wild boar and the ancient pig confirms that Asian introgression in modern Iberian pigs has not existed or has been negligible. In contrast, the Guatemalan Creole pig clusters apart from the Iberian pig genome, likely due to introgression from international breeds.