RESUMO
The use of hybridization capture has enabled a massive upscaling in sample sizes for ancient DNA studies, allowing the analysis of hundreds of skeletal remains or sediments in single studies. Nevertheless, demands in throughput continue to grow, and hybridization capture has become a limiting step in sample preparation due to the large consumption of reagents, consumables and time. Here, we explored the possibility of improving the economics of sample preparation via multiplex capture, that is, the hybridization capture of pools of double-indexed ancient DNA libraries. We demonstrate that this strategy is feasible, at least for small genomic targets such as mitochondrial DNA, if the annealing temperature is increased and PCR cycles are limited in post-capture amplification to avoid index swapping by jumping PCR, which manifests as cross-contamination in resulting sequence data. We also show that the reamplification of double-indexed libraries to PCR plateau before or after hybridization capture can sporadically lead to small, but detectable cross-contamination even if libraries are amplified in separate reactions. We provide protocols for both manual capture and automated capture in 384-well format that are compatible with single- and multiplex capture and effectively suppress cross-contamination and artefact formation. Last, we provide a simple computational method for quantifying cross-contamination due to index swapping in double-indexed libraries, which we recommend using for routine quality checks in studies that are sensitive to cross-contamination.
Assuntos
DNA Antigo , Genômica , DNA Antigo/análise , DNA Mitocondrial/genética , Hibridização de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodosRESUMO
The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.
Assuntos
Restos Mortais/metabolismo , Impressões Digitais de DNA/métodos , DNA Antigo/análise , Genética Forense , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , DNA Antigo/isolamento & purificação , Humanos , Guerra da Coreia , II Guerra MundialRESUMO
SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast screening method based on direct RT-qPCR to detect SARS-CoV-2 in single or pooled gargle lavages ("mouthwashes"). This method detects individuals with large viral loads (Ct≤29) and we use it to test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches can be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics.
Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19 , Programas de Rastreamento , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/genética , HumanosRESUMO
It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.
Assuntos
DNA Antigo , DNA de Cadeia Simples/genética , Biblioteca Gênica , Análise de Sequência de DNA/métodos , Automação , Sequência de BasesRESUMO
BACKGROUND: The identification of bona fide microbial taxa in microbiomes derived from ancient and historical samples is complicated by the unavoidable mixture between DNA from ante- and post-mortem microbial colonizers. One possibility to distinguish between these sources of microbial DNA is querying for the presence of age-associated degradation patterns typical of ancient DNA (aDNA). The presence of uracils, resulting from cytosine deamination, has been detected ubiquitously in aDNA retrieved from diverse sources, and used as an authentication criterion. Here, we employ a library preparation method that separates molecules that carry uracils from those that do not for a set of samples that includes Neandertal remains, herbarium specimens and archaeological plant remains. RESULTS: We show that sequencing DNA libraries enriched in molecules carrying uracils effectively amplifies age associated degradation patterns in microbial mixtures of ancient and historical origin. This facilitates the discovery of authentic ancient microbial taxa in cases where degradation patterns are difficult to detect due to large sequence divergence in microbial mixtures. Additionally, the relative enrichment of taxa in the uracil enriched fraction can help to identify bona fide ancient microbial taxa that could be missed using a more targeted approach. CONCLUSIONS: Our experiments show, that in addition to its use in enriching authentic endogenous DNA of organisms of interest, the selective enrichment of damaged DNA molecules can be a valuable tool in the discovery of ancient microbial taxa.
Assuntos
Bactérias/classificação , DNA Antigo/análise , Fósseis/microbiologia , Análise de Sequência de DNA/métodos , Uracila/química , Animais , Bactérias/genética , DNA Antigo/química , DNA Bacteriano/genética , Mineração de Dados , Biblioteca Gênica , Metagenômica , Microbiota , Homem de Neandertal/microbiologia , Plantas/microbiologiaRESUMO
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.
Assuntos
Osso e Ossos/química , DNA Antigo/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Extração em Fase Sólida/métodos , Dente/química , Fósseis , Biblioteca Gênica , Sedimentos Geológicos/química , Humanos , Imãs , Dióxido de Silício/química , Extração em Fase Sólida/instrumentaçãoRESUMO
North Africa is a key region for understanding human history, but the genetic history of its people is largely unknown. We present genomic data from seven 15,000-year-old modern humans, attributed to the Iberomaurusian culture, from Morocco. We find a genetic affinity with early Holocene Near Easterners, best represented by Levantine Natufians, suggesting a pre-agricultural connection between Africa and the Near East. We do not find evidence for gene flow from Paleolithic Europeans to Late Pleistocene North Africans. The Taforalt individuals derive one-third of their ancestry from sub-Saharan Africans, best approximated by a mixture of genetic components preserved in present-day West and East Africans. Thus, we provide direct evidence for genetic interactions between modern humans across Africa and Eurasia in the Pleistocene.
Assuntos
População Negra/genética , Evolução Molecular , Genoma Humano , Genoma Mitocondrial , África Subsaariana , África do Norte , Animais , DNA Antigo , Feminino , Fluxo Gênico , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Oriente Médio , Polimorfismo de Nucleotídeo Único , População BrancaRESUMO
By at least 45,000 years before present, anatomically modern humans had spread across Eurasia [1-3], but it is not well known how diverse these early populations were and whether they contributed substantially to later people or represent early modern human expansions into Eurasia that left no surviving descendants today. Analyses of genome-wide data from several ancient individuals from Western Eurasia and Siberia have shown that some of these individuals have relationships to present-day Europeans [4, 5] while others did not contribute to present-day Eurasian populations [3, 6]. As contributions from Upper Paleolithic populations in Eastern Eurasia to present-day humans and their relationship to other early Eurasians is not clear, we generated genome-wide data from a 40,000-year-old individual from Tianyuan Cave, China, [1, 7] to study his relationship to ancient and present-day humans. We find that he is more related to present-day and ancient Asians than he is to Europeans, but he shares more alleles with a 35,000-year-old European individual than he shares with other ancient Europeans, indicating that the separation between early Europeans and early Asians was not a single population split. We also find that the Tianyuan individual shares more alleles with some Native American groups in South America than with Native Americans elsewhere, providing further support for population substructure in Asia [8] and suggesting that this persisted from 40,000 years ago until the colonization of the Americas. Our study of the Tianyuan individual highlights the complex migration and subdivision of early human populations in Eurasia.
Assuntos
DNA Antigo/análise , Genoma Humano , Migração Humana , Arqueologia , Variação Biológica da População , China , Humanos , Masculino , FilogeniaRESUMO
Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.
Assuntos
DNA Antigo/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Hominidae/classificação , Hominidae/genética , Animais , Cavernas , DNA Antigo/análise , DNA Mitocondrial/análise , Europa (Continente) , Fósseis , Sedimentos Geológicos/química , Análise de Sequência de DNARESUMO
A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago. An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals. However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history.
Assuntos
Hominidae/genética , Filogenia , Alelos , Animais , DNA Mitocondrial/genética , Fósseis , Genoma Mitocondrial/genética , Hominidae/classificação , Masculino , Homem de Neandertal/classificação , Homem de Neandertal/genética , Alinhamento de Sequência , EspanhaRESUMO
Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.
Assuntos
Contaminação por DNA , Fósseis , Paleodontologia , Bactérias , Osso e Ossos , Descontaminação/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Paleodontologia/métodos , DenteRESUMO
We present the high-quality genome sequence of a â¼45,000-year-old modern human male from Siberia. This individual derives from a population that lived before-or simultaneously with-the separation of the populations in western and eastern Eurasia and carries a similar amount of Neanderthal ancestry as present-day Eurasians. However, the genomic segments of Neanderthal ancestry are substantially longer than those observed in present-day individuals, indicating that Neanderthal gene flow into the ancestors of this individual occurred 7,000-13,000 years before he lived. We estimate an autosomal mutation rate of 0.4 × 10(-9) to 0.6 × 10(-9) per site per year, a Y chromosomal mutation rate of 0.7 × 10(-9) to 0.9 × 10(-9) per site per year based on the additional substitutions that have occurred in present-day non-Africans compared to this genome, and a mitochondrial mutation rate of 1.8 × 10(-8) to 3.2 × 10(-8) per site per year based on the age of the bone.
Assuntos
Fósseis , Genoma Humano/genética , Alelos , Animais , Cromossomos Humanos Par 12/genética , Dieta , Evolução Molecular , Humanos , Hibridização Genética/genética , Masculino , Dados de Sequência Molecular , Taxa de Mutação , Homem de Neandertal/genética , Filogenia , Densidade Demográfica , Dinâmica Populacional , Análise de Componente Principal , Análise de Sequência de DNA , SibériaRESUMO
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.
Assuntos
Fósseis , Genoma Mitocondrial/genética , Hominidae/classificação , Hominidae/genética , Filogenia , Animais , Teorema de Bayes , Sequência Consenso/genética , Citosina/metabolismo , DNA Mitocondrial/genética , Desaminação , Fêmur/anatomia & histologia , Fêmur/metabolismo , Hominidae/anatomia & histologia , Dados de Sequência Molecular , Homem de Neandertal/genética , EspanhaRESUMO
Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.
Assuntos
Animais Domésticos , Animais Selvagens , Encéfalo/metabolismo , Expressão Gênica , Antígeno AC133 , Animais , Animais Domésticos/genética , Animais Domésticos/metabolismo , Animais Selvagens/genética , Animais Selvagens/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Comportamento Animal , Cães , Glicoproteínas/genética , Glicoproteínas/metabolismo , Cobaias , Peptídeos/genética , Peptídeos/metabolismo , Coelhos , Ratos , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismo , Sus scrofa , LobosRESUMO
Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals.
Assuntos
Evolução Molecular , Perfilação da Expressão Gênica , RNA Mensageiro/genética , Animais , Humanos , Filogenia , Análise de Componente Principal , Cromossomo X/genéticaRESUMO
Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.