TICI: a taxon-independent community index for eDNA-based ecological health assessment.

Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Ancient DNA provides insights into 4,000 years of resource economy across Greenland.

The success and failure of past cultures across the Arctic was tightly coupled to the ability of past peoples to exploit the full range of resources available to them. There is substantial evidence for the hunting of birds, caribou and seals in prehistoric Greenland. However, the extent to which these communities relied on fish and cetaceans is understudied because of taphonomic processes that affect how these taxa are presented in the archaeological record. To address this, we analyse DNA from bulk bone samples from 12 archaeological middens across Greenland covering the Palaeo-Inuit, Norse and Neo-Inuit culture. We identify an assemblage of 42 species, including nine fish species and five whale species, of which the bowhead whale (Balaena mysticetus) was the most commonly detected. Furthermore, we identify a new haplotype in caribou (Rangifer tarandus), suggesting the presence of a distinct lineage of (now extinct) dwarfed caribou in Greenland 3,000 years ago.

Author Correction: Early Pastoral Economies and Herding Transitions in Eastern Eurasia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Early Pastoral Economies and Herding Transitions in Eastern Eurasia.

While classic models for the emergence of pastoral groups in Inner Asia describe mounted, horse-borne herders sweeping across the Eurasian Steppes during the Early or Middle Bronze Age (ca. 3000-1500 BCE), the actual economic basis of many early pastoral societies in the region is poorly characterized. In this paper, we use collagen mass fingerprinting and ancient DNA analysis of some of the first stratified and directly dated archaeofaunal assemblages from Mongolia's early pastoral cultures to undertake species identifications of this rare and highly fragmented material. Our results provide evidence for livestock-based, herding subsistence in Mongolia during the late 3rd and early 2nd millennia BCE. We observe no evidence for dietary exploitation of horses prior to the late Bronze Age, ca. 1200 BCE - at which point horses come to dominate ritual assemblages, play a key role in pastoral diets, and greatly influence pastoral mobility. In combination with the broader archaeofaunal record of Inner Asia, our analysis supports models for widespread changes in herding ecology linked to the innovation of horseback riding in Central Asia in the final 2nd millennium BCE. Such a framework can explain key broad-scale patterns in the movement of people, ideas, and material culture in Eurasian prehistory.

Species-level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization.

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding-based biodiversity studies is gaining popularity as a noninvasive, time-efficient, and cost-effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under-surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA-related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species-level assignment) and universal (broad taxonomic group with genus/family-level assignment) approaches obtained from replicates treated with the optimal and a low-performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose-nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false-negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose-nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol-chloroform-isoamyl for successful implementation of eDNA multi-marker metabarcoding surveys.

Combined use of eDNA metabarcoding and video surveillance for the assessment of fish biodiversity.

Monitoring communities of fish is important for the management and sustainability of fisheries and marine ecosystems. Baited remote underwater video systems (BRUVs) are among the most effective nondestructive techniques for sampling bony fishes and elasmobranchs (sharks, rays, and skates). However, BRUVs sample visually conspicuous biota; hence, some taxa are undersampled or not recorded at all. We compared the diversity of fishes characterized using BRUVs with diversity detected via environmental DNA (eDNA) metabarcoding. We sampled seawater and captured BRUVs imagery at 48 locales that included reef and seagrass beds inside and outside a marine reserve (Jurien Bay in Western Australia). Eighty-two fish genera from 13 orders were detected, and the community of fishes described using eDNA and BRUVs combined yielded >30% more generic richness than when either method was used alone. Rather than detecting a homogenous genetic signature, the eDNA assemblages mirrored the BRUVs' spatial explicitness; differentiation of taxa between seagrass and reef was clear despite the relatively small geographical scale of the study site (∼35 km2 ). Taxa that were not sampled by one approach, due to limitations and biases intrinsic to the method, were often detected with the other. Therefore, using BRUVs and eDNA in concert provides a more holistic view of vertebrate marine communities across habitats. Both methods are noninvasive, which enhances their potential for widespread implementation in the surveillance of marine ecosystems.

Pollutant emissions and environmental assessment of ethyl 3-ethoxybutyrate, a potential renewable fuel.

Renewable and bio-based transportation fuel sources can lower the life-cycle greenhouse gas emissions from vehicles. We present an initial assessment of ethyl 3-ethoxybutyrate (EEB) as a biofuel in terms of its performance as a fuel oxygenate and its persistence in the environment. EEB can be produced from ethanol and poly-3-hydroxybutyrate, a bacterial storage polymer that can be produced from non-food biomass and other organic feedstocks. Physicochemical properties of EEB and fuel-relevant properties of EEB-gasoline blends were measured, emissions of criteria pollutants from EEB as a gasoline additive in a production vehicle were evaluated, and fate and persistence of EEB in the environment were estimated. EEB solubility in water was 25.8 g/L, its Kow was 1.8, and its Henry's Law constant was 1.04 × 10(-5) atm-m(3)/mole. The anti-knock index values for 5 and 20 % v/v EEB-gasoline blends were 91.6 and 91.9, respectively. Reductions in fuel economy were consistent with the level of oxygenation, and criteria emissions were met by the vehicle operated over the urban dynamometer driving cycle (FTP 75). Predicted environmental persistence ranged from 15 to 30 days which indicates that EEB is not likely to be a persistent organic pollutant. In combination, these results suggest a high potential for the use of EEB as a renewable fuel source.

Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled.

Metagenomic analyses of bacteria on human hairs: a qualitative assessment for applications in forensic science.

BACKGROUND: Mammalian hairs are one of the most ubiquitous types of trace evidence collected in the course of forensic investigations. However, hairs that are naturally shed or that lack roots are problematic substrates for DNA profiling; these hair types often contain insufficient nuclear DNA to yield short tandem repeat (STR) profiles. Whilst there have been a number of initial investigations evaluating the value of metagenomics analyses for forensic applications (e.g. examination of computer keyboards), there have been no metagenomic evaluations of human hairs-a substrate commonly encountered during forensic practice. This present study attempts to address this forensic capability gap, by conducting a qualitative assessment into the applicability of metagenomic analyses of human scalp and pubic hair. RESULTS: Forty-two DNA extracts obtained from human scalp and pubic hairs generated a total of 79,766 reads, yielding 39,814 reads post control and abundance filtering. The results revealed the presence of unique combinations of microbial taxa that can enable discrimination between individuals and signature taxa indigenous to female pubic hairs. Microbial data from a single co-habiting couple added an extra dimension to the study by suggesting that metagenomic analyses might be of evidentiary value in sexual assault cases when other associative evidence is not present. CONCLUSIONS: Of all the data generated in this study, the next-generation sequencing (NGS) data generated from pubic hair held the most potential for forensic applications. Metagenomic analyses of human hairs may provide independent data to augment other forensic results and possibly provide association between victims of sexual assault and offender when other associative evidence is absent. Based on results garnered in the present study, we believe that with further development, bacterial profiling of hair will become a valuable addition to the forensic toolkit.

Quantitative real-time PCR in aDNA research.

Quantitative real-time PCR (qPCR) is a technique that is widely used in the field of ancient DNA (aDNA). Quantitative PCR can be used to optimize aDNA extraction methodologies, to detect PCR inhibition, and to quantify aDNA libraries for use in high-throughput sequencing. In this chapter, we outline factors that need to be considered when developing efficient SYBR Green qPCR assays. We describe how to setup qPCR standards of known copy number and provide some useful tips regarding interpretation of qPCR data generated from aDNA templates.