A workflow for accurate metabarcoding using nanopore MinION sequencing

1. Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short‐read high‐throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low‐cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. 2. To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full‐length consensus DNA barcodes for a bulk mock sample of 50 aquatic invertebrate species with at least 15% genetic distance to each other. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build error‐corrected consensus sequences. A newly developed Python pipeline, ASHURE, was used for data processing, consensus building, clustering, and taxonomic assignment of the resulting reads. 3. Our pipeline achieved median accuracies of up to 99.3% for long concatemeric reads (>45 barcodes) and successfully identified all 50 species in the mock community. The use of RCA was integral for increasing consensus accuracy but was also the most time‐consuming step of the laboratory workflow. Most concatemeric reads were skewed towards a shorter read length range with a median read length of up to 1262bp. 4. Our study demonstrates that Nanopore sequencing can be used for metabarcoding, but exploration of other isothermal amplification procedures to improve consensus accuracy is recommended

Riparian forests can mitigate warming and ecological degradation of agricultural headwater streams

1. Riparian forests are commonly advocated as a key management option to mitigate the effects of agriculture on headwater stream biodiversity and ecosystem func tions. However, the benefits of riparian forests might be reduced by uninterrupted catchment-scale pollution. 2. We studied the effects of riparian land use on multiple ecological endpoints in head water streams in an agricultural landscape. We studied stream habitat characteristics, water temperature and algal accrual, and macrophyte, benthic macroinvertebrate and fish communities in 11 paired forested and open agricultural headwater stream reaches that differed in their extent of riparian forest cover but had similar water quality. 3. Hydromorphological habitat quality was higher in forested reaches than in open reaches. Riparian forest had a strong effect on the summer water temperature regime, with maximum and mean water temperatures and temperature variation in forested reaches substantially lower than in open reaches. 4. Macrophyte communities differed between forested and open reaches. The mean abundance of bryophytes was higher in forested reaches but the difference to open reaches was only marginally significant, whereas graminoids were significantly more abundant in open reaches. Within-stream dissimilarity of benthic macroinvertebrate community structure was significantly related to the difference in riparian land use between reach pairs. The relative DNA sequence abundance of pollution-sensitive Ephemeroptera, Plecoptera, and Trichoptera species tended to be higher in forested reaches than in open reaches. Finally, fish densities were not significantly different be tween forested and open reaches, although densities were higher in forested reaches. 5. This unequivocal evidence for the ecological benefits of forested riparian reaches in agricultural headwater streams suggests that riparian forest can partly mitigate the adverse impacts of agricultural diffuse pollution on biota. The strong effect of forests on stream water temperature suggest that riparian forest could also miti gate harmful effects on headwater stream biodiversity and ecosystem functions of the predicted more frequent high summer temperatures

Strategies for sample labelling and library preparation in DNA metabarcoding studies.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample-specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample-specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one-step PCR, two-step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications

Advancing the use of molecular methods for routine freshwater macroinvertebrate biomonitoring : the need for calibration experiments

Over the last decade, steady advancements have been made in the use of DNA-based methods for detection of species in a wide range of ecosystems. This progress has culminated in molecular monitoring methods being employed for the detection of several species for enforceable management purposes of endangered, invasive, and illegally harvested species worldwide. However, the routine application of DNA-based methods to monitor whole communities (typically a metabarcoding approach) in order to assess the status of ecosystems continues to be limited. In aquatic ecosystems, the limited use is particularly true for macroinvertebrate communities. As part of the DNAqua-Net consortium, a structured discussion was initiated with the aim to identify potential molecular methods for freshwater macroinvertebrate community assessment and identify important knowledge gaps for their routine application. We focus on three complementary DNA sources that can be metabarcoded: 1) DNA from homogenised samples (bulk DNA), 2) DNA extracted from sample preservative (fixative DNA), and 3) environmental DNA (eDNA) from water or sediment. We provide a brief overview of metabarcoding macroinvertebrate communities from each DNA source and identify challenges for their application to routine monitoring. To advance the utilisation of DNA-based monitoring for macroinvertebrates, we propose an experimental design template for a series of methodological calibration tests. The template compares sources of DNA with the goal of identifying the effects of molecular processing steps on precision and accuracy. Furthermore, the same samples will be morphologically analysed, which will enable the benchmarking of molecular to traditional processing approaches. In doing so we hope to highlight pathways for the development of DNA-based methods for the monitoring of freshwater macroinvertebrates

Why we need sustainable networks bridging countries, disciplines, cultures and generations for Aquatic Biomonitoring 2.0: A Perspective Derived From the DNAqua-Net COST Action

Aquatic biomonitoring has become an essential task in Europe and many other regions as a consequence of strong anthropogenic pressures affecting the health of lakes, rivers, oceans and groundwater. A typical assessment of the environmental quality status, such as it is required by European but also North American and other legislation, relies on matching the composition of assemblages of organisms identified using morphological criteria present in aquatic ecosystems to those expected in the absence of anthropogenic pressures. Through decade-long and difficult intercalibration exercises among networks of regulators and scientists in European countries, a pragmatic biomonitoring approach was developed and adopted, which now produces invaluable information. Nonetheless, this approach is based on several hundred different protocols, making it susceptible to issues with comparability, scale and resolution. Furthermore, data acquisition is often slow due to a lack of taxonomic experts for many taxa and regions and time-consuming morphological identification of organisms. High-throughput genetic screening methods such as (e)DNA metabarcoding have been proposed as a possible solution to these shortcomings. Such "next-generation biomonitoring", also termed "biomonitoring 2.0", has many advantages over the traditional approach in terms of speed, comparability and costs. It also creates the potential to include new bioindicators and thereby further improves the assessment of aquatic ecosystem health. However, several major conceptual and technological challenges still hinder its implementation into legal and regulatory frameworks. Academic scientists sometimes tend to overlook legal or socioeconomic constraints, which regulators have to consider on a regular basis. Moreover, quantification of species abundance or biomass remains a significant bottleneck to releasing the full potential of these approaches. Here, we highlight the main challenges for next-generation aquatic biomonitoring and outline principles and good practicCOST - European Cooperation in Science and Technology(CA15219). COST Action DNAqua-Net (CA15219), supported by the COST (European Cooperation in Science and Technology) programm

COI Primer bind

Reference OTU sequence alignments of the HCO / LCO Folmer primer binding regions barcoding region for 15 relevant freshwater taxa (Downloaded with PrimerMiner February 2015, 26 bp trimming applied to sequences before clustering

PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

Abstract 13 1) DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker 14 region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of 15 optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical 16 importance. However, no tool for obtaining and processing of reference sequence data in bulk that serve as a backbone for 17 primer design is currently available. 18 2) We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and 19 NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units 20 (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner 21 offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in 22 another available software for that purpose, ecoPCR. 23 3) We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater 24 invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to 25 increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. 26 Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using 27 PrimerMiner

Data from: PrimerMiner: an R package for development and in silico validation of DNA metabarcoding primers

1. DNA metabarcoding is a powerful tool to assess biodiversity by amplifying and sequencing a standardized gene marker region. Its success is often limited due to variable binding sites that introduce amplification biases. Thus the development of optimized primers for communities or taxa under study in a certain geographic region and/or ecosystems is of critical importance. However, no tool for obtaining and processing of reference sequence data in bulk that can serve as a backbone for primer design is currently available. 2. We developed the R package PrimerMiner, which batch downloads DNA barcode gene sequences from BOLD and NCBI databases for specified target taxonomic groups and then applies sequence clustering into operational taxonomic units (OTUs) to reduce biases introduced by the different number of available sequences per species. Additionally, PrimerMiner offers functionalities to evaluate primers in silico, which are in our opinion more realistic then the strategy employed in another available software for that purpose, ecoPCR. 3. We used PrimerMiner to download cytochrome c oxidase subunit I (COI) sequences for 15 important freshwater invertebrate groups, relevant for ecosystem assessment. By processing COI markers from both databases, we were able to increase the amount of reference data 249-fold on average, compared to using complete mitochondrial genomes alone. Furthermore, we visualized the generated OTU sequence alignments and describe how to evaluate primers in silico using PrimerMiner. 4. With PrimerMiner we provide a useful tool to obtain relevant sequence data for targeted primer development and evaluation. The OTU based reference alignments generated with PrimerMiner can be used for manual primer design, or processed with bioinformatic tools for primer development

Metabarcoding pipeline and scripts

Full metabarcoding pipeline and R scripts to produce figures and statistical analysis in the paper. Includes intermediate scripts, always use the script with the latest version number