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

Genomic data reveals strong differentiation and reduced genetic diversity in island golden eagle populations

Understanding population structure and the extent and distribution of genetic diversity are recognised as central issues in endangered species research, with broad implications for effective conservation management. Advances in whole genome sequencing (WGS) techniques provide greater resolution of genome-wide genetic diversity and inbreeding. Subspecies of golden eagles (Aquila chrysaetos) in Scotland (A. c. chrysaetos) and Japan (A. c. japonica) are endangered; it is therefore important to understand genetic diversity and inbreeding of these small island populations to increase the chances of conservation success. We investigated this using WGS data from golden eagles in Scotland, continental Europe, Japan, and the USA. Following determination of population genetic structure, analysis of heterozygosity and nucleotide diversity revealed reduced levels of genetic diversity together with runs of homozygosity (ROH), suggesting evidence of inbreeding due to recent shared parental ancestry in the island populations. These results highlight the need to consider genetic reinforcement of small isolated golden eagle populations from neighbouring outbred populations, alongside existing efforts to boost population size through within-island conservation translocations and captive breeding programmes

Group Dynamics of Zebra and Wildebeest in a Woodland Savanna: Effects of Predation Risk and Habitat Density

.Using generalized linear models, we examined the relative importance of habitat type (differing in vegetation density), probability of encountering lion (based on utilization distribution of all individual lions in the reserve), and season in predicting group size and composition. We found that only in open scrub habitat, group size for both ungulate species increased with the probability of encountering lion. Group composition differed between the two species and was driven by habitat selection as well as predation risk. For both species, composition of groups was, however, dominated by males in open scrub habitats, irrespective of the probability of encountering lion.Distribution patterns of wildebeest and zebra groups at the landscape level directly support the theoretical and empirical evidence from a range of taxa predicting that grouping is favored in open habitats and when predation risk is high. Group composition reflected species-specific social, physiological and foraging constraints, as well as the importance of predation risk. Avoidance of high resource open scrub habitat by females can lead to loss of foraging opportunities, which can be particularly costly in areas such as KGR, where this resource is limited. Thus, landscape-level grouping dynamics are species specific and particular to the composition of the group, arising from a tradeoff between maximizing resource selection and minimizing predation risk

DART Mass spectrometry as a potential tool for the differentiation of captive-bred and wild lion bones.

In recent years lion bones have been legally traded internationally to Asian markets from captive bred sources in South Africa. There are also indications of increasing instances of illegal international trade in wild lion bones. The existence of parallel captive and wild supplies of lion bone are a cause of law enforcement concern regarding the potential for the laundering of illegally sourced bones through legal trade, and present a problem for the assessment of the conservation impact of wild lion bone trade due to the difficulty of determining what market-share wild and captive-bred lion bones account for. Captive-bred and wild lion bone are visually indistinguishable and no reliable method currently exists for distinguishing them. We present a preliminary study that explores the use of DART mass spectrometry as a method to differentiate between captive-bred and wild lion bones. We find that DART is able to differentiate between a batch of captive-bred South African lion bone and a batch of wild lion bone and suggest that DART mass spectrometry shows strong potential as a tool for the regulation and investigation of lion bone trade. Further testing is needed to prove the suitability of this technique. Therefore, we suggest that further research focuses on testing the capability of DART to differentiate between contemporary wild and captive-bred lion bone originating from South Africa, and attempts to identify chemical markers in bone that can be used as indicators of captive-bred origin

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

Investigating the origins of ivory recovered in the United Kingdom

Over recent years, mounting pressure has been placed on countries to assess their role in the ivory trade, with a view to tackling the rapidly declining numbers of elephants, due to poaching. The United Kingdom has been identified as a large re-exporter of ivory. Despite much of this trade being reported as legal or antique ivory, such provision of ivory to meet demand is known to fuel illegal markets and provide trade routes for modern ivory sales. Aside from ivory species and age, further analysis to evaluate geographic provenance, can inform where an elephant had lived, and so identify a source region or population where poaching occurred. The purpose of this study was to determine the age and species of ivory objects surrendered or seized in the UK and assess their likely geographic provenance through comparison of results from mitochondrial DNA and stable isotope analysis to publicly accessible georeferenced African elephant databases. The results demonstrated that the objects tested from an airport seizure were modern and matched existing haplotypes allowing for regional geographic inferences (supported by both techniques) to be obtained for most of these objects. In contrast, antique and modern ivory was detected amongst the amnesty objects, and several new mtDNA haplotypes were identified. Regional geographic inferences were achieved for some but not all of the objects tested. Our findings show this combination of methods provides a wealth of information which, could provide insight into targeted elephant populations and assist in disrupting international wildlife trade networks

Evaluation of approaches for identifying population informative markers from high density SNP Chips

<p>Abstract</p> <p>Background</p> <p>Genetic markers can be used to identify and verify the origin of individuals. Motivation for the inference of ancestry ranges from conservation genetics to forensic analysis. High density assays featuring Single Nucleotide Polymorphism (SNP) markers can be exploited to create a reduced panel containing the most informative markers for these purposes. The objectives of this study were to evaluate methods of marker selection and determine the minimum number of markers from the BovineSNP50 BeadChip required to verify the origin of individuals in European cattle breeds. Delta, Wright's F_ST, Weir & Cockerham's F_STand PCA methods for population differentiation were compared. The level of informativeness of each SNP was estimated from the breed specific allele frequencies. Individual assignment analysis was performed using the ranked informative markers. Stringency levels were applied by log-likelihood ratio to assess the confidence of the assignment test.</p> <p>Results</p> <p>A 95% assignment success rate for the 384 individually genotyped animals was achieved with < 80, < 100, < 140 and < 200 SNP markers (with increasing stringency threshold levels) across all the examined methods for marker selection. No further gain in power of assignment was achieved by sampling in excess of 200 SNP markers. The marker selection method that required the lowest number of SNP markers to verify the animal's breed origin was Wright's F_ST(60 to 140 SNPs depending on the chosen degree of confidence). Certain breeds required fewer markers (< 100) to achieve 100% assignment success. In contrast, closely related breeds require more markers (~200) to achieve > 95% assignment success. The power of assignment success, and therefore the number of SNP markers required, is dependent on the levels of genetic heterogeneity and pool of samples considered.</p> <p>Conclusions</p> <p>While all SNP selection methods produced marker panels capable of breed identification, the power of assignment varied markedly among analysis methods. Thus, with effective exploration of available high density genetic markers, a diagnostic panel of highly informative markers can be produced.</p