Nanopore sequencing in non-human forensic genetics

The past decade has seen a rapid expansion of non-human forensic genetics coinciding with the development of 2nd and 3rd generation DNA sequencing technologies. Nanopore sequencing is one such technology that offers massively parallel sequencing at a fraction of the capital cost of other sequencing platforms. The application of nanopore sequencing to species identification has already been widely demonstrated in biomonitoring studies and has significant potential for non-human forensic casework, particularly in the area of wildlife forensics. This review examines nanopore sequencing technology and assesses its potential applications, advantages and drawbacks for use in non-human forensics, alongside other next-generation sequencing platforms and as a possible replacement to Sanger sequencing. We assess the specific challenges of sequence error rate and the standardisation of consensus sequence production, before discussing recent progress in the validation of nanopore sequencing for use in forensic casework. We conclude that nanopore sequencing may be able to play a considerable role in the future of non-human forensic genetics, especially for applications to wildlife law enforcement within emerging forensic laboratories

DNA analysis of Late Glacial mammal remains

Ich habe im Rahmen meiner Dimpolarbeit DNA von 10 modernen und 67 subfossilen Lemmingknochen (modern bis 25.200 Jahre kal. BP) aus dem noerdlichen Ural (Russland) extrahiert, amplifiziert und diese mittels 454 FLX ”next-generation” Pyrosequencing (ROCHE) sequenziert. Auf Markov Chain Monte Carlo (MCMC) basierende Bayesian Analysen und Approximate Bayesian Computation (ABC) Analysen wurden benutzt um die zeitliche demographische Veraederung der Lemmingpopulation zu rekonstruieren. Der Plot der effektiven Populationsgroesse (Ne) zeigt ein deutliches ”Bottleneck” (d.h. einen drastischen Abfall) der zeitlich mit der Klimaerwaermung im Boelling/Alleroed zusammenfaellt. Halsbandlemminge sind spezialisiert auf kaltes und sehr trockenes Klima. Diese hochspezialisierten oekologischen Anforderungen und paleontologische Untersuchungen unterstreichen die Glaubwuerdigkeit der genetischen Resultate weiters. Um den Einfluss von ”gene flow” bzw. Migration auf meine Daten zu bestimmen habe ich ein Haplotypennetzwerk mit zusaetzlichen modernen Sequenzen errechnet. Die Ergebnisse dieser Analyse und die Interpretation des temporalen Netzwerke deuten auf eine erhoehte Migrationsrate nach dem Bottleneck hin. Aus publizierten Studien an modernen Lemmingen ist dieses Phenomaen (der Anstieg der Migrationrate wenn die Populationsdichte sinkt) bereits bekannt. Diese Erkenntnisse stehen im Einklang mit den erhaltenen Resultaten. Analysen mit zusaetzlichen subfossilen Lemmingen aus einer weiteren Fundstelle (Yangana-Pe-4) deuten auf das Vorhandensein einer Kontaktzone der regionalen Haplotypengruppen (Yamal und Pymva Shor) hin. Dies zeigt, dass Migration heute nur regional eingeschraenkt stattfindet. Die Ergebnisse dieser Arbeit unterstreichen die Nuetzlichkeit von temporaerem DNA sampling (moderne und subfossile DNA) um die Auswirkungen von Klimaeveraenderungen auf Saeugetierpopulationen zu studieren.I used temporal DNA sampling to study the demographic changes in the collared lemming (Dicrostonyx torquatus) from Pymva Shor in the northern Ural. I successfully extracted, amplified and sequenced DNA from 10 modern and 77 ancient lemming remains. Using makrov chain monte carlo (MCMC) based bayesian inference and approximate bayesian computation (ABC) I was able to show that the abrupt warming event at the Bolling/Allerod interstadial correlated with the timing of a severe population decline in the collared lemming. Given the strong adaptation to dry and cold climate of Dicrostonyx this finding suggests a coherence between the population bottleneck and this abrupt warming event. Using additional modern DNA sequences I showed that there are signs for gene flow in my sampled data, which is coherent with the assumption of a higher migration rate following a strong population decline in lemmings. A contact zone between individuals bearing the Yamal haplotype and the major haplotype from Pymva Shor was found in (approximately 1000 year old) samples from Yangana-Pe-4 and thus, suggesting modern gene flow only on a small geographical scale. My study underlines the usefulness of temporal ancient DNA data for a proper understanding of the effects of climate change on mammal populations

High-throughput sequencing for community analysis: the promise of DNA barcoding to uncover diversity, relatedness, abundances and interactions in spider communities

Large-scale studies on community ecology are highly desirable but often difficult to accomplish due to the considerable investment of time, labor and, money required to characterize richness, abundance, relatedness, and interactions. Nonetheless, such large-scale perspectives are necessary for understanding the composition, dynamics, and resilience of biological communities. Small invertebrates play a central role in ecosystems, occupying critical positions in the food web and performing a broad variety of ecological functions. However, it has been particularly difficult to adequately characterize communities of these animals because of their exceptionally high diversity and abundance. Spiders in particular fulfill key roles as both predator and prey in terrestrial food webs and are hence an important focus of ecological studies. In recent years, large-scale community analyses have benefitted tremendously from advances in DNA barcoding technology. High-throughput sequencing (HTS), particularly DNA metabarcoding, enables community-wide analyses of diversity and interactions at unprecedented scales and at a fraction of the cost that was previously possible. Here, we review the current state of the application of these technologies to the analysis of spider communities. We discuss amplicon-based DNA barcoding and metabarcoding for the analysis of community diversity and molecular gut content analysis for assessing predator-prey relationships. We also highlight applications of the third generation sequencing technology for long read and portable DNA barcoding. We then address the development of theoretical frameworks for community-level studies, and finally highlight critical gaps and future directions for DNA analysis of spider communities

Developmental validation of Oxford Nanopore Technology MinION sequence data and the NGSpeciesID bioinformatic pipeline for forensic genetic species identification

Species identification of non-human biological evidence through DNA nucleotide sequencing is routinely used for forensic genetic analysis to support law enforcement. The gold standard for forensic genetics is conventional Sanger sequencing; however, this is gradually being replaced by high-throughput sequencing (HTS) approaches which can generate millions of individual reads in a single experiment. HTS sequencing, which now dominates molecular biology research, has already been demonstrated for use in a number of forensic genetic analysis applications, including species identification. However, the generation of HTS data to date requires expensive equipment and is cost-effective only when large numbers of samples are analysed simultaneously. The Oxford Nanopore Technologies (ONT) MinION™ is an affordable and small footprint DNA sequencing device with the potential to quickly deliver reliable and cost effective data. However, there has been no formal validation of forensic species identification using high-throughput (deep read) sequence data from the MinION making it currently impractical for many wildlife forensic end-users. Here, we present a MinION deep read sequence data validation study for species identification. First, we tested whether the clustering-based bioinformatics pipeline NGSpeciesID can be used to generate an accurate consensus sequence for species identification. Second, we systematically evaluated the read variation distribution around the generated consensus sequences to understand what confidence we have in the accuracy of the resulting consensus sequence and to determine how to interpret individual sample results. Finally, we investigated the impact of differences between the MinION consensus and Sanger control sequences on correct species identification to understand the ability and accuracy of the MinION consensus sequence to differentiate the true species from the next most similar species. This validation study establishes that ONT MinION sequence data used in conjunction with the NGSpeciesID pipeline can produce consensus DNA sequences of sufficient accuracy for forensic genetic species identification

Diversity of selected toll-like receptor genes in cheetahs (Acinonyx jubatus) and African leopards (Panthera pardus pardus).

BackgroundThe growing world population amplifies the anthropogenic impact on wildlife globally. With shrinking habitats, wild populations are being pushed to co-exist in close proximity to humans, leading to an increased threat of infectious disease. Therefore, understanding the immune system of a species is key to assess its resilience in a changing environment. The innate immunity system (IIS) is the body’s first line of defense against pathogens. High variability in IIS-genes, such as the toll-like receptor (TLR) genes, appears to be associated with resistance to infectious diseases. However, few studies have investigated diversity in TLR genes in non-model organisms and drawn conclusions for the conservation of vulnerable species. Large predators are threatened globally, and their populations increasingly have been declining over the last decades. Big cats, such as leopards (Panthera pardus) and cheetahs (Acinonyx jubatus) are no exception to this trend and are listed as ‘vulnerable’ by the International Union for Conservation of Nature (IUCN) including several subspecies, e.g., A. j. venaticus and P. p. melas, that already face extinction. To better understand vulnerability in terms of immune genetic diversity in the two sympatric occurring species, we compared selected TLR genes (TLR2, TLR4, TLR6 and TLR8) between modern African leopards (P. p. pardus) and Southern African cheetahs (A. j. jubatus). ResultsOur study supports the previously detected high genetic diversity in African leopards and confirms genetic impoverishment in Southern African cheetahs. Despite notable differences, both species share some haplotypic similarities in the investigated TLRs. Moreover, our historic cheetah samples from all five subspecies showed levels of genetic diversity comparable to modern African leopards. By including historic cheetahs and samples from all known subspecies, we put the observed IIS diversity into an evolutionary context.ConclusionThe genetic diversity in the investigated TLR genes in modern Southern African cheetahs and in historic cheetahs is low compared to African leopards. However, according to previous studies, the low immune genetic diversity might not yet affect the health of this cheetah subspecies. Compared to historic cheetah data and other subspecies, a more recent population decline might explain the observed genetic impoverishment of TLR genes in modern Southern African cheetahs.<br/

Co-occurrence of ecologically similar species of Hawaiian spiders reveals critical early phase of adaptive radiation

Background: The processes through which populations originate and diversify ecologically in the initial stages of adaptive radiation are little understood because we lack information on critical steps of early divergence. A key question is, at what point do closely related species interact, setting the stage for competition and ecological specialization? The Hawaiian Islands provide an ideal system to explore the early stages of adaptive radiation because the islands span ages from 0.5–5 Mya. Hawaiian spiders in the genus Tetragnatha have undergone adaptive radiation, with one lineage (“spiny legs�) showing four different ecomorphs (green, maroon, large brown, small brown); one representative of each ecomorph is generally found at any site on the older islands. Given that the early stages of adaptive radiation are characterized by allopatric divergence between populations of the same ecomorph, the question is, what are the steps towards subsequent co-occurrence of different ecomorphs? Using a transcriptome-based exon capture approach, we focus on early divergence among close relatives of the green ecomorph to understand processes associated with co-occurrence within the same ecomorph at the early stages of adaptive radiation. Results: The major outcomes from the current study are first that closely related species within the same green ecomorph of spiny leg Tetragnatha co-occur on the same single volcano on East Maui, and second that there is no evidence of genetic admixture between these ecologically equivalent species. Further, that multiple genetic lineages exist on a single volcano on Maui suggests that there are no inherent dispersal barriers and that the observed limited distribution of taxa reflects competitive exclusion. Conclusions: The observation of co-occurrence of ecologically equivalent species on the young volcano of Maui provides a missing link in the process of adaptive radiation between the point when recently divergent species of the same ecomorph occur in allopatry, to the point where different ecomorphs co-occur at a site, as found throughout the older islands. More importantly, the ability of close relatives of the same ecomorph to interact, without admixture, may provide the conditions necessary for ecological divergence and independent evolution of ecomorphs associated with adaptive radiation

Integrating Multiple Lines of Evidence into Historical Biogeography Hypothesis Testing: A Bison bison Case Study

One of the grand goals of historical biogeography is to understand how and why species’ population sizes and distributions change over time. Multiple types of data drawn from disparate fields, combined into a single modelling framework, are necessary to document changes in a species’s demography and distribution, and to determine the drivers responsible for change. Yet truly integrated approaches are challenging and rarely performed. Here, we discuss a modelling framework that integrates spatio-temporal fossil data, ancient DNA, palaeoclimatological reconstructions, bioclimatic envelope modelling and coalescence models in order to statistically test alternative hypotheses of demographic and potential distributional changes for the iconic American bison (Bison bison). Using different assumptions about the evolution of the bioclimatic niche, we generate hypothetical distributional and demographic histories of the species. We then test these demographic models by comparing the genetic signature predicted by serial coalescence against sequence data derived from subfossils and modern populations. Our results supported demographic models that include both climate and human-associated drivers of population declines. This synthetic approach, integrating palaeoclimatology, bioclimatic envelopes, serial coalescence, spatio-temporal fossil data and heterochronous DNA sequences, improves understanding of species’ historical biogeography by allowing consideration of both abiotic and biotic interactions at the population level

The Tetragnatha kauaiensis genome sheds light on the origins of genomic novelty in spider

Spiders (Araneae) have a diverse spectrum of morphologies, behaviors, and physiologies. Attempts to understand the genomic-basis of this diversity are often hindered by their large, heterozygous, and AT-rich genomes with high repeat content resulting in highly fragmented, poor-quality assemblies. As a result, the key attributes of spider genomes, including gene family evolution, repeat content, and gene function, remain poorly understood. Here, we used Illumina and Dovetail Chicago technologies to sequence the genome of the long-jawed spider Tetragnatha kauaiensis, producing an assembly distributed along 3,925 scaffolds with an N50 of ∼2 Mb. Using comparative genomics tools, we explore genome evolution across available spider assemblies. Our findings suggest that the previously reported and vast genome size variation in spiders is linked to the different representation and number of transposable elements. Using statistical tools to uncover gene-family level evolution, we find expansions associated with the sensory perception of taste, immunity, and metabolism. In addition, we report strikingly different histories of chemosensory, venom, and silk gene families, with the first two evolving much earlier, affected by the ancestral whole genome duplication in Arachnopulmonata (∼450 Ma) and exhibiting higher numbers. Together, our findings reveal that spider genomes are highly variable and that genomic novelty may have been driven by the burst of an ancient whole genome duplication, followed by gene family and transposable element expansion