py_ped_sim - A flexible forward genetic simulator for complex family pedigree analysis.

Background: Large-scale family pedigrees are commonly used across medical, evolutionary, and forensic genetics. These pedigrees are tools for identifying genetic disorders, tracking evolutionary patterns, and establishing familial relationships via forensic genetic identification. However, there is a lack of software to accurately simulate different pedigree structures along with genomes corresponding to those individuals in a family pedigree. This limits simulation-based evaluations of methods that use pedigrees. Results: We have developed a python command-line-based tool called py_ped_sim that facilitates the simulation of pedigree structures and the genomes of individuals in a pedigree. py_ped_sim represents pedigrees as directed acyclic graphs, enabling conversion between standard pedigree formats and integration with the forward population genetic simulator, SLiM. Notably, py_ped_sim allows the simulation of varying numbers of offspring for a set of parents, with the capacity to shift the distribution of sibship sizes over generations. We additionally add simulations for events of misattributed paternity, which offers a way to simulate half-sibling relationships. We validated the accuracy of our software by simulating genomes onto diverse family pedigree structures, showing that the estimated kinship coefficients closely approximated expected values. Conclusions: py_ped_sim is a user-friendly and open-source solution for simulating pedigree structures and conducting pedigree genome simulations. It empowers medical, forensic, and evolutionary genetics researchers to gain deeper insights into the dynamics of genetic inheritance and relatedness within families.

Stable isotopes show Homo sapiens dispersed into cold steppes ~45,000 years ago at Ilsenhöhle in Ranis, Germany.

The spread of Homo sapiens into new habitats across Eurasia ~45,000 years ago and the concurrent disappearance of Neanderthals represents a critical evolutionary turnover in our species' history. 'Transitional' technocomplexes, such as the Lincombian-Ranisian-Jerzmanowician (LRJ), characterize the European record during this period but their makers and evolutionary significance have long remained unclear. New evidence from Ilsenhöhle in Ranis, Germany, now provides a secure connection of the LRJ to H. sapiens remains dated to ~45,000 years ago, making it one of the earliest forays of our species to central Europe. Using many stable isotope records of climate produced from 16 serially sampled equid teeth spanning ~12,500 years of LRJ and Upper Palaeolithic human occupation at Ranis, we review the ability of early humans to adapt to different climate and habitat conditions. Results show that cold climates prevailed across LRJ occupations, with a temperature decrease culminating in a pronounced cold excursion at ~45,000-43,000 cal BP. Directly dated H. sapiens remains confirm that humans used the site even during this very cold phase. Together with recent evidence from the Initial Upper Palaeolithic, this demonstrates that humans operated in severe cold conditions during many distinct early dispersals into Europe and suggests pronounced adaptability.

Chronological and genetic analysis of an Upper Palaeolithic female infant burial from Borsuka Cave, Poland.

Six infant human teeth and 112 animal tooth pendants from Borsuka Cave were identified as the oldest burial in Poland. However, uncertainties around the dating and the association of the teeth to the pendants have precluded their association with an Upper Palaeolithic archaeological industry. Using <67 mg per tooth, we combined dating and genetic analyses of two human teeth and six herbivore tooth pendants to address these questions. Our interdisciplinary approach yielded informative results despite limited sampling material, and high levels of degradation and contamination. Our results confirm the Palaeolithic origin of the human remains and herbivore pendants, and permit us to identify the infant as female and discuss the association of the assemblage with different Palaeolithic industries. This study exemplifies the progress that has been made toward minimally destructive methods and the benefits of integrating methods to maximize data retrieval from precious but highly degraded and contaminated prehistoric material.

Review: Computational analysis of human skeletal remains in ancient DNA and forensic genetics.

Degraded DNA is used to answer questions in the fields of ancient DNA (aDNA) and forensic genetics. While aDNA studies typically center around human evolution and past history, and forensic genetics is often more concerned with identifying a specific individual, scientists in both fields face similar challenges. The overlap in source material has prompted periodic discussions and studies on the advantages of collaboration between fields toward mutually beneficial methodological advancements. However, most have been centered around wet laboratory methods (sampling, DNA extraction, library preparation, etc.). In this review, we focus on the computational side of the analytical workflow. We discuss limitations and considerations to consider when working with degraded DNA. We hope this review provides a framework to researchers new to computational workflows for how to think about analyzing highly degraded DNA and prompts an increase of collaboration between the forensic genetics and aDNA fields.

Ancient human DNA recovered from a Palaeolithic pendant.

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals1 who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited2-5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000-25,000 years. Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.

Genetic insights into the social organization of Neanderthals.

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

Quantifying and reducing cross-contamination in single- and multiplex hybridization capture of ancient DNA.

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.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

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.

Pleistocene sediment DNA reveals hominin and faunal turnovers at Denisova Cave.

Denisova Cave in southern Siberia is the type locality of the Denisovans, an archaic hominin group who were related to Neanderthals1-4. The dozen hominin remains recovered from the deposits also include Neanderthals5,6 and the child of a Neanderthal and a Denisovan7, which suggests that Denisova Cave was a contact zone between these archaic hominins. However, uncertainties persist about the order in which these groups appeared at the site, the timing and environmental context of hominin occupation, and the association of particular hominin groups with archaeological assemblages5,8-11. Here we report the analysis of DNA from 728 sediment samples that were collected in a grid-like manner from layers dating to the Pleistocene epoch. We retrieved ancient faunal and hominin mitochondrial (mt)DNA from 685 and 175 samples, respectively. The earliest evidence for hominin mtDNA is of Denisovans, and is associated with early Middle Palaeolithic stone tools that were deposited approximately 250,000 to 170,000 years ago; Neanderthal mtDNA first appears towards the end of this period. We detect a turnover in the mtDNA of Denisovans that coincides with changes in the composition of faunal mtDNA, and evidence that Denisovans and Neanderthals occupied the site repeatedly-possibly until, or after, the onset of the Initial Upper Palaeolithic at least 45,000 years ago, when modern human mtDNA is first recorded in the sediments.

Unearthing Neanderthal population history using nuclear and mitochondrial DNA from cave sediments.

Bones and teeth are important sources of Pleistocene hominin DNA, but are rarely recovered at archaeological sites. Mitochondrial DNA (mtDNA) has been retrieved from cave sediments but provides limited value for studying population relationships. We therefore developed methods for the enrichment and analysis of nuclear DNA from sediments and applied them to cave deposits in western Europe and southern Siberia dated to between 200,000 and 50,000 years ago. We detected a population replacement in northern Spain about 100,000 years ago, which was accompanied by a turnover of mtDNA. We also identified two radiation events in Neanderthal history during the early part of the Late Pleistocene. Our work lays the ground for studying the population history of ancient hominins from trace amounts of nuclear DNA in sediments.

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.