Unravelling reference bias in ancient DNA datasets.

MOTIVATION: The alignment of sequencing reads is a critical step in the characterization of ancient genomes. However, reference bias and spurious mappings pose a significant challenge, particularly as cutting-edge wet lab methods generate datasets that push the boundaries of alignment tools. Reference bias occurs when reference alleles are favoured over alternative alleles during mapping, whereas spurious mappings stem from either contamination or when endogenous reads fail to align to their correct position. Previous work has shown that these phenomena are correlated with read length but a more thorough investigation of reference bias and spurious mappings for ancient DNA has been lacking. Here, we use a range of empirical and simulated palaeogenomic datasets to investigate the impacts of mapping tools, quality thresholds, and reference genome on mismatch rates across read lengths. RESULTS: For these analyses, we introduce AMBER, a new bioinformatics tool for assessing the quality of ancient DNA mapping directly from BAM-files and informing on reference bias, read length cut-offs and reference selection. AMBER rapidly and simultaneously computes the sequence read mapping bias in the form of the mismatch rates per read length, cytosine deamination profiles at both CpG and non-CpG sites, fragment length distributions, and genomic breadth and depth of coverage. Using AMBER, we find that mapping algorithms and quality threshold choices dictate reference bias and rates of spurious alignment at different read lengths in a predictable manner, suggesting that optimized mapping parameters for each read length will be a key step in alleviating reference bias and spurious mappings. AVAILABILITY AND IMPLEMENTATION: AMBER is available for noncommercial use on GitHub (https://github.com/tvandervalk/AMBER.git). Scripts used to generate and analyse simulated datasets are available on Github (https://github.com/sdolenz/refbias_scripts).

An endothelial regulatory module links blood pressure regulation with elite athletic performance.

The control of transcription is crucial for homeostasis in mammals. A previous selective sweep analysis of horse racing performance revealed a 19.6 kb candidate regulatory region 50 kb downstream of the Endothelin3 (EDN3) gene. Here, the region was narrowed to a 5.5 kb span of 14 SNVs, with elite and sub-elite haplotypes analyzed for association to racing performance, blood pressure and plasma levels of EDN3 in Coldblooded trotters and Standardbreds. Comparative analysis of human HiCap data identified the span as an enhancer cluster active in endothelial cells, interacting with genes relevant to blood pressure regulation. Coldblooded trotters with the sub-elite haplotype had significantly higher blood pressure compared to horses with the elite performing haplotype during exercise. Alleles within the elite haplotype were part of the standing variation in pre-domestication horses, and have risen in frequency during the era of breed development and selection. These results advance our understanding of the molecular genetics of athletic performance and vascular traits in both horses and humans.

Equine herpesvirus 4 infected domestic horses associated with Sintashta spoke-wheeled chariots around 4,000 years ago.

Equine viral outbreaks have disrupted the socio-economic life of past human societies up until the late 19th century and continue to be of major concern to the horse industry today. With a seroprevalence of 60-80 per cent, equine herpesvirus 4 (EHV-4) is the most common horse pathogen on the planet. Yet, its evolutionary history remains understudied. Here, we screen the sequenced data of 264 archaeological horse remains to detect the presence of EHV-4. We recover the first ancient EHV-4 genome with 4.2× average depth-of-coverage from a specimen excavated in the Southeastern Urals and dated to the Early Bronze Age period, approximately 3,900 years ago. The recovery of an EHV-4 virus outside the upper respiratory tract not only points to an animal particularly infected but also highlights the importance of post-cranial bones in pathogen characterisation. Bayesian phylogenetic reconstruction provides a minimal time estimate for EHV-4 diversification to around 4,000 years ago, a time when modern domestic horses spread across the Central Asian steppes together with spoke-wheeled Sintashta chariots, or earlier. The analyses also considerably revise the diversification time of the two EHV-4 subclades from the 16th century based solely on modern data to nearly a thousand years ago. Our study paves the way for a robust reconstruction of the history of non-human pathogens and their impact on animal health.

Isotopic biographies reveal horse rearing and trading networks in medieval London.

This paper reports a high-resolution isotopic study of medieval horse mobility, revealing their origins and in-life mobility both regionally and internationally. The animals were found in an unusual horse cemetery site found within the City of Westminster, London, England. Enamel strontium, oxygen, and carbon isotope analysis of 15 individuals provides information about likely place of birth, diet, and mobility during the first approximately 5 years of life. Results show that at least seven horses originated outside of Britain in relatively cold climates, potentially in Scandinavia or the Western Alps. Ancient DNA sexing data indicate no consistent sex-specific mobility patterning, although three of the five females came from exceptionally highly radiogenic regions. Another female with low mobility is suggested to be a sedentary broodmare. Our results provide direct and unprecedented evidence for a variety of horse movement and trading practices in the Middle Ages and highlight the importance of international trade in securing high-quality horses for medieval London elites.

Tracking population structure and phenology through time using ancient genomes from waterlogged white oak wood.

Whole genome characterizations of crop plants based on ancient DNA have provided unique keys for a better understanding of the evolutionary origins of modern cultivars, the pace and mode of selection underlying their adaptation to new environments and the production of phenotypes of interest. Although forests are among the most biologically rich ecosystems on earth and represent a fundamental resource for human societies, no ancient genome sequences have been generated for trees. This contrasts with the generation of multiple ancient reference genomes for important crops. Here, we sequenced the first ancient tree genomes using two white oak wood remains from Germany dating to the Last Little Ice Age (15th century CE, 7.3× and 4.0×) and one from France dating to the Bronze Age (1700 BCE, 3.4×). We assessed the underlying species and identified one medieval remains as a hybrid between two common oak species (Quercus robur and Q. petraea) and the other two remains as Q. robur. We found that diversity at the global genome level had not changed over time. However, exploratory analyses suggested that a reduction of diversity took place at different time periods. Finally, we determined the timing of leaf unfolding for ancient trees for the first time. The study extends the application of ancient wood beyond the classical proxies of dendroclimatology, dendrochronology, dendroarchaeology and dendroecology, thereby enhancing resolution of inferences on the responses of forest ecosystems to past environmental changes, epidemics and silvicultural practices.

The history of the Coast Salish "woolly dogs".

DNA and traditional knowledge reveal the history of an extinct dog bred for its wool.

Interdisciplinary evidence for early domestic horse exploitation in southern Patagonia.

The introduction of domestic horses transformed Indigenous societies across the grasslands of Argentina, leading to the emergence of specialized horse cultures across the Southern Cone. However, the dynamics of this introduction are poorly chronicled by historic records. Here, we apply archaeozoological and biomolecular techniques to horse remains from the site of Chorrillo Grande 1 in southern Argentina. Osteological and taphonomic analyses suggest that horses were pastorally managed and used for food by Aónikenk/Tehuelche hunter-gatherers before the onset of permanent European settlement, as early as the mid-17th century. DNA-based sex identifications suggest consumption of both male and female horses, while ceramic residue also shows use of guanaco products. Sequential isotope analyses on horse dentition reveal an origin in southern Patagonia and movement of these animals between the Río Coig and Río Gallegos basins. These results reinforce emerging evidence for rapid Indigenous-mediated dispersal of horses in the Americas and demonstrate that horses catalyzed rapid economic and social transformations.

Imputed genomes of historical horses provide insights into modern breeding.

Historical genomes can provide important insights into recent genomic changes in horses, especially the development of modern breeds. In this study, we characterized 8.7 million genomic variants from a panel of 430 horses from 73 breeds, including newly sequenced genomes from 20 Clydesdales and 10 Shire horses. We used this modern genomic variation to impute the genomes of four historically important horses, consisting of publicly available genomes from 2 Przewalski's horses, 1 Thoroughbred, and a newly sequenced Clydesdale. Using these historical genomes, we identified modern horses with higher genetic similarity to those in the past and unveiled increased inbreeding in recent times. We genotyped variants associated with appearance and behavior to uncover previously unknown characteristics of these important historical horses. Overall, we provide insights into the history of Thoroughbred and Clydesdale breeds and highlight genomic changes in the endangered Przewalski's horse following a century of captive breeding.

Refining the evolutionary tree of the horse Y chromosome.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski's horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Improving the extraction of ancient Yersinia pestis genomes from the dental pulp.

Ancient DNA preserved in the dental pulp offers the opportunity to characterize the genome of some of the deadliest pathogens in human history. However, while DNA capture technologies help, focus sequencing efforts, and therefore, reduce experimental costs, the recovery of ancient pathogen DNA remains challenging. Here, we tracked the kinetics of ancient Yersinia pestis DNA release in solution during a pre-digestion of the dental pulp. We found that most of the ancient Y. pestis DNA is released within 60 min at 37°C in our experimental conditions. We recommend a simple pre-digestion as an economical procedure to obtain extracts enriched in ancient pathogen DNA, as longer digestion times release other types of templates, including host DNA. Combining this procedure with DNA capture, we characterized the genome sequences of 12 ancient Y. pestis bacteria from France dating to the second pandemic outbreaks of the 17th and 18th centuries Common Era.

DNA methylation-based profiling of horse archaeological remains for age-at-death and castration.

Age profiling of archaeological bone assemblages can inform on past animal management practices, but is limited by the fragmentary nature of the fossil record and the lack of universal skeletal markers for age. DNA methylation clocks offer new, albeit challenging, alternatives for estimating the age-at-death of ancient individuals. Here, we take advantage of the availability of a DNA methylation clock based on 31,836 CpG sites and dental age markers in horses to assess age predictions in 84 ancient remains. We evaluate our approach using whole-genome sequencing data and develop a capture assay providing reliable estimates for only a fraction of the cost. We also leverage DNA methylation patterns to assess castration practice in the past. Our work opens for a deeper characterization of past husbandry and ritual practices and holds the potential to reveal age mortality profiles in ancient societies, once extended to human remains.

The genomic history and global expansion of domestic donkeys.

Donkeys transformed human history as essential beasts of burden for long-distance movement, especially across semi-arid and upland environments. They remain insufficiently studied despite globally expanding and providing key support to low- to middle-income communities. To elucidate their domestication history, we constructed a comprehensive genome panel of 207 modern and 31 ancient donkeys, as well as 15 wild equids. We found a strong phylogeographic structure in modern donkeys that supports a single domestication in Africa ~5000 BCE, followed by further expansions in this continent and Eurasia and ultimately returning to Africa. We uncover a previously unknown genetic lineage in the Levant ~200 BCE, which contributed increasing ancestry toward Asia. Donkey management involved inbreeding and the production of giant bloodlines at a time when mules were essential to the Roman economy and military.

mapDATAge: a ShinyR package to chart ancient DNA data through space and time.

SUMMARY: Ancient DNA datasets are increasingly difficult to visualize for users lacking computational experience. Here, we describe mapDATAge, which aims to provide user-friendly automated modules for the interactive mapping of allele, haplogroup and/or ancestry distributions through space and time. mapDATAge enhances collaborative data sharing while assisting the assessment and reporting of spatiotemporal patterns of genetic changes. AVAILABILITY AND IMPLEMENTATION: mapDATAge is a Shiny R application designed for exploring spatiotemporal patterns in ancient DNA data through a graphical user interface. It is freely available under GNU Public License in Github: https://github.com/xuefenfei712/mapDATAge. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Radiocarbon and genomic evidence for the survival of Equus Sussemionus until the late Holocene.

The exceptionally rich fossil record available for the equid family has provided textbook examples of macroevolutionary changes. Horses, asses, and zebras represent three extant subgenera of Equus lineage, while the Sussemionus subgenus is another remarkable Equus lineage ranging from North America to Ethiopia in the Pleistocene. We sequenced 26 archaeological specimens from Northern China in the Holocene that could be assigned morphologically and genetically to Equus ovodovi, a species representative of Sussemionus. We present the first high-quality complete genome of the Sussemionus lineage, which was sequenced to 13.4× depth of coverage. Radiocarbon dating demonstrates that this lineage survived until ~3500 years ago, despite continued demographic collapse during the Last Glacial Maximum and the great human expansion in East Asia. We also confirmed the Equus phylogenetic tree and found that Sussemionus diverged from the ancestor of non-caballine equids ~2.3-2.7 million years ago and possibly remained affected by secondary gene flow post-divergence. We found that the small genetic diversity, rather than enhanced inbreeding, limited the species' chances of survival. Our work adds to the growing literature illustrating how ancient DNA can inform on extinction dynamics and the long-term resilience of species surviving in cryptic population pockets.

Assessing the impact of USER-treatment on hyRAD capture applied to ancient DNA.

Ancient DNA preservation in subfossil specimens provides a unique opportunity to retrieve genetic information from the past. As ancient DNA extracts are generally dominated by molecules originating from environmental microbes, capture techniques are often used to economically retrieve orthologous sequence data at the population scale. Post-mortem DNA damage, especially the deamination of cytosine residues into uracils, also considerably inflates sequence error rates unless ancient DNA extracts are treated with the USER enzymatic mix prior to library construction. While both approaches have recently gained popularity in ancient DNA research, the impact of USER-treatment on capture efficacy still remains untested. In this study, we applied hyRAD capture to eight ancient equine subfossil specimens from France (1st-17th century CE), including horses, donkeys and their first-generation mule hybrids. We found that USER-treatment could reduce capture efficacy and introduce significant experimental bias. It differentially affected the size distribution of on-target templates following capture with two distinct hyRAD probe sets in a manner that was not driven by differences in probe sizes and DNA methylation levels. Finally, we recovered unbalanced proportions of donkey-specific and horse-specific alleles in mule capture sequence data, due to the combined effects of USER-treatment, probe sets and reference bias. Our work demonstrates that while USER-treatment can improve the quality of ancient DNA sequence data, it can also significantly affect hyRAD capture outcomes, introducing bias in the sequence data that is difficult to predict based on simple molecular probe features. Such technical batch effects may prove easier to model and correct for using capture with synthetic probes of controlled sizes and diversity content.