Detection of chromosomal aneuploidy in ancient genomes.

Ancient DNA is a valuable tool for investigating genetic and evolutionary history that can also provide detailed profiles of the lives of ancient individuals. In this study, we develop a generalised computational approach to detect aneuploidies (atypical autosomal and sex chromosome karyotypes) in the ancient genetic record and distinguish such karyotypes from contamination. We confirm that aneuploidies can be detected even in low-coverage genomes ( ~ 0.0001-fold), common in ancient DNA. We apply this method to ancient skeletal remains from Britain to document the first instance of mosaic Turner syndrome (45,X0/46,XX) in the ancient genetic record in an Iron Age individual sequenced to average 9-fold coverage, the earliest known incidence of an individual with a 47,XYY karyotype from the Early Medieval period, as well as individuals with Klinefelter (47,XXY) and Down syndrome (47,XY, + 21). Overall, our approach provides an accessible and automated framework allowing for the detection of individuals with aneuploidies, which extends previous binary approaches. This tool can facilitate the interpretation of burial context and living conditions, as well as elucidate past perceptions of biological sex and people with diverse biological traits.

Yersinia pestis genomes reveal plague in Britain 4000 years ago.

Extinct lineages of Yersinia pestis, the causative agent of the plague, have been identified in several individuals from Eurasia between 5000 and 2500 years before present (BP). One of these, termed the 'LNBA lineage' (Late Neolithic and Bronze Age), has been suggested to have spread into Europe with human groups expanding from the Eurasian steppe. Here, we show that the LNBA plague was spread to Europe's northwestern periphery by sequencing three Yersinia pestis genomes from Britain, all dating to ~4000 cal BP. Two individuals were from an unusual mass burial context in Charterhouse Warren, Somerset, and one individual was from a single burial under a ring cairn monument in Levens, Cumbria. To our knowledge, this represents the earliest evidence of LNBA plague in Britain documented to date. All three British Yersinia pestis genomes belong to a sublineage previously observed in Bronze Age individuals from Central Europe that had lost the putative virulence factor yapC. This sublineage is later found in Eastern Asia ~3200 cal BP. While the severity of the disease is currently unclear, the wide geographic distribution within a few centuries suggests substantial transmissibility.

Dual ancestries and ecologies of the Late Glacial Palaeolithic in Britain.

Genetic investigations of Upper Palaeolithic Europe have revealed a complex and transformative history of human population movements and ancestries, with evidence of several instances of genetic change across the European continent in the period following the Last Glacial Maximum (LGM). Concurrent with these genetic shifts, the post-LGM period is characterized by a series of significant climatic changes, population expansions and cultural diversification. Britain lies at the extreme northwest corner of post-LGM expansion and its earliest Late Glacial human occupation remains unclear. Here we present genetic data from Palaeolithic human individuals in the United Kingdom and the oldest human DNA thus far obtained from Britain or Ireland. We determine that a Late Upper Palaeolithic individual from Gough's Cave probably traced all its ancestry to Magdalenian-associated individuals closely related to those from sites such as El Mirón Cave, Spain, and Troisième Caverne in Goyet, Belgium. However, an individual from Kendrick's Cave shows no evidence of having ancestry related to the Gough's Cave individual. Instead, the Kendrick's Cave individual traces its ancestry to groups who expanded across Europe during the Late Glacial and are represented at sites such as Villabruna, Italy. Furthermore, the individuals differ not only in their genetic ancestry profiles but also in their mortuary practices and their diets and ecologies, as evidenced through stable isotope analyses. This finding mirrors patterns of dual genetic ancestry and admixture previously detected in Iberia but may suggest a more drastic genetic turnover in northwestern Europe than in the southwest.

Reconstructing double-stranded DNA fragments on a single-molecule level reveals patterns of degradation in ancient samples.

Extensive manipulations involved in the preparation of DNA samples for sequencing have hitherto made it impossible to determine the precise structure of double-stranded DNA fragments being sequenced, such as the presence of blunt ends, single-stranded overhangs, or single-strand breaks. We here describe MatchSeq, a method that combines single-stranded DNA library preparation from diluted DNA samples with computational sequence matching, allowing the reconstruction of double-stranded DNA fragments on a single-molecule level. The application of MatchSeq to Neanderthal DNA, a particularly complex source of degraded DNA, reveals that 1- or 2-nt overhangs and blunt ends dominate the ends of ancient DNA molecules and that short gaps exist, which are predominantly caused by the loss of individual purines. We further show that deamination of cytosine to uracil occurs in both single- and double-stranded contexts close to the ends of molecules, and that single-stranded parts of DNA fragments are enriched in pyrimidines. MatchSeq provides unprecedented resolution for interrogating the structures of fragmented double-stranded DNA and can be applied to fragmented double-stranded DNA isolated from any biological source. The method relies on well-established laboratory techniques and can easily be integrated into routine data generation. This possibility is shown by the successful reconstruction of double-stranded DNA fragments from previously published single-stranded sequence data, allowing a more comprehensive characterization of the biochemical properties not only of ancient DNA but also of cell-free DNA from human blood plasma, a clinically relevant marker for the diagnosis and monitoring of disease.

A genetic analysis of the Gibraltar Neanderthals.

The Forbes' Quarry and Devil's Tower partial crania from Gibraltar are among the first Neanderthal remains ever found. Here, we show that small amounts of ancient DNA are preserved in the petrous bones of the 2 individuals despite unfavorable climatic conditions. However, the endogenous Neanderthal DNA is present among an overwhelming excess of recent human DNA. Using improved DNA library construction methods that enrich for DNA fragments carrying deaminated cytosine residues, we were able to sequence 70 and 0.4 megabase pairs (Mbp) nuclear DNA of the Forbes' Quarry and Devil's Tower specimens, respectively, as well as large parts of the mitochondrial genome of the Forbes' Quarry individual. We confirm that the Forbes' Quarry individual was a female and the Devil's Tower individual a male. We also show that the Forbes' Quarry individual is genetically more similar to the ∼120,000-y-old Neanderthals from Scladina Cave in Belgium (Scladina I-4A) and Hohlenstein-Stadel Cave in Germany, as well as to a ∼60,000- to 70,000-y-old Neanderthal from Russia (Mezmaiskaya 1), than to a ∼49,000-y-old Neanderthal from El Sidrón (El Sidrón 1253) in northern Spain and other younger Neanderthals from Europe and western Asia. This suggests that the Forbes' Quarry fossil predates the latter Neanderthals. The preservation of archaic human DNA in the warm coastal climate of Gibraltar, close to the shores of Africa, raises hopes for the future recovery of archaic human DNA from regions in which climatic conditions are less than optimal for DNA preservation.

Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing.

DNA preserved in ancient bones, teeth and sediments is typically highly fragmented and present only in minute amounts. Here, we provide a highly versatile silica-based DNA extraction protocol that enables the retrieval of short (≥35 bp) or even ultrashort (≥25 bp) DNA fragments from such material with minimal carryover of substances that inhibit library preparation for high-throughput sequencing. DNA extraction can be performed with either silica spin columns, which offer the most convenient choice for manual DNA extraction, or silica-coated magnetic particles. The latter allow a substantial cost reduction as well as automation on liquid-handling systems. This protocol update replaces a now-outdated version that was published 11 years ago, before high-throughput sequencing technologies became widely available. It has been thoroughly optimized to provide the highest DNA yields from highly degraded samples, as well as fast and easy handling, requiring not more than ~15 min of hands-on time per sample.

Extending the spectrum of DNA sequences retrieved from ancient bones and teeth.

The number of DNA fragments surviving in ancient bones and teeth is known to decrease with fragment length. Recent genetic analyses of Middle Pleistocene remains have shown that the recovery of extremely short fragments can prove critical for successful retrieval of sequence information from particularly degraded ancient biological material. Current sample preparation techniques, however, are not optimized to recover DNA sequences from fragments shorter than ∼35 base pairs (bp). Here, we show that much shorter DNA fragments are present in ancient skeletal remains but lost during DNA extraction. We present a refined silica-based DNA extraction method that not only enables efficient recovery of molecules as short as 25 bp but also doubles the yield of sequences from longer fragments due to improved recovery of molecules with single-strand breaks. Furthermore, we present strategies for monitoring inefficiencies in library preparation that may result from co-extraction of inhibitory substances during DNA extraction. The combination of DNA extraction and library preparation techniques described here substantially increases the yield of DNA sequences from ancient remains and provides access to a yet unexploited source of highly degraded DNA fragments. Our work may thus open the door for genetic analyses on even older material.

Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

A mitochondrial genome sequence of a hominin from Sima de los Huesos.

Excavations of a complex of caves in the Sierra de Atapuerca in northern Spain have unearthed hominin fossils that range in age from the early Pleistocene to the Holocene. One of these sites, the 'Sima de los Huesos' ('pit of bones'), has yielded the world's largest assemblage of Middle Pleistocene hominin fossils, consisting of at least 28 individuals dated to over 300,000 years ago. The skeletal remains share a number of morphological features with fossils classified as Homo heidelbergensis and also display distinct Neanderthal-derived traits. Here we determine an almost complete mitochondrial genome sequence of a hominin from Sima de los Huesos and show that it is closely related to the lineage leading to mitochondrial genomes of Denisovans, an eastern Eurasian sister group to Neanderthals. Our results pave the way for DNA research on hominins from the Middle Pleistocene.

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

Object-based analysis of astroglial reaction and astrocyte subtype morphology after ischemic brain injury.

The astrocytic response to ischemic brain injury is characterized by specific alterations of glial cell morphology and function. Various studies described both beneficial and detrimental aspects of activated astrocytes, suggesting the existence of different subtypes. We investigated this issue using a novel object-based approach to study characteristics of astrogliosis after stroke. Spontaneously hypertensive rats received permanent middle cerebral artery occlusion. After 96 h, brain specimens were removed, fixed and stained for GFAP, glutamine synthetase (GS), S100Beta and Musashi1 (Msh1). Three regions of interest were defined (contralateral hemisphere, ipsilateral remote zone and infarct border zone), and confocal stacks were acquired (n=5 biological with each n=4 technical replicates). The stacks were background-corrected and colocalization between the selected markers and GFAP was determined using an automated thresholding algorithm. The fluorescence and colocalization channels were then converted into 3D-objects using both intensity and volume as filters to ultimately determine the final volumes of marker expression and colocalization, as well as the morphological changes of astrocyte process arborisation. We found that both S100Beta and Msh1 determined the same GFAP-positive astroglial cell population albeit the cellular compartments differed. GFAP stained most of the astrocyte processes and is hence suitable for the analysis of qualitative characteristics of astrogliosis. Due to its peri-nuclear localization, Msh1 is appropriate to estimate the total number of astrocytes even in regions with severe reactive astrogliosis. GS expression in GFAP-positive astrocytes was high in the remote zone and low at the infarct border, indicating the existence of astrocyte subclasses.